national institute of science and technology in stem cell

National Institute of Science and Technology Edital Nº 15/2008

MCT/CNPq/FNDCT/CAPES/FAPEMIG/FAPERJ/FAPESP

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IINNCCTTCC

Coordinator: Roberto Passetto Falcão Vice-Coordinator: Dimas Tadeu Covas

Coordinating Institution:

Faculdade de Medicina e Hemocentro de Ribeirão Preto, Universidade de São Paulo

Associated Research Laboratories and Groups •Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da USP. •Laboratório de Morfofisiologia Molecular e do Desenvolvimento da Faculdade de Zootecnia

e Engenharia de Alimentos de Pirassunga, USP •Centro de Transplantes de Medula Óssea do Hospital das Clínicas da Faculdade de

Medicina de Ribeirão Preto da USP •Departamento de Cirurgia, setor de Anatomia dos Animais Domésticos e Silvestres da

Faculdade de Medicina Veterinária da USP •Instituto de Ciências Biomédicas da UFRJ •Laboratórios de Terapia Celular do Hemocentro de Hemocentro de Ribeirão Preto –

HCFMRP-USP •Laboratório de Biologia Celular do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Biologia Molecular do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Transferência Gênica do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Criobiologia I e II do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Biotecnologia do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de HLA do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Anemias Hereditárias do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Genética Molecular do Hemocentro de Ribeirão Preto – HCFMRP-USP •Laboratório de Citometria de Fluxo do Hemocentro de Ribeirão Preto – HCFMRP-USP •Centro Químico de Proteínas do Hemocentro de Ribeirão Preto – HCFMRP-USP •Centro de Primatologia do Instituto Evandro Chagas de Belém, Pará. •Departamento de Genética e Biologia Evolutiva do Instituto de Biociências – USP •Laboratório de Genética da Faculdade de Medicina de Ribeirão Preto – USP •Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos – USP •Departamento de Clínica Médica, Divisão de Imunologia Clínica - FMRP-USP

Principal Researchers • Dimas Tadeu Covas – Fundação Hemocentro de Ribeirão Preto • Eduardo Magalhães Rego – Faculdade de Medicina de Ribeirão Preto/USP • Flavio Vieira Meirelles - Faculdade de Zootecnia e Engenharia de Alimentos • Júlio Cesar Voltarelli - Faculdade de Medicina de Ribeirão Preto/USP • João Palermo Neto – Faculdade de Medicina Veterinária e Zootecnia/USP • Klena Sarges Marruaz da Silva–Centro de Primatas do Institudo Evandro Chagas–Belém-PA

• Lewis Joel Greene - Faculdade de Medicina de Ribeirão Preto/USP • Lygia da Veiga Pereira - Instituto de Biociências/USP • Maria Angélica Miglino - Faculdade de Medicina Veterinária e Zootecnia/USP • Roberto Passetto Falcão - Faculdade de Medicina de Ribeirão Preto/USP • Stevens Kastrup Rehen - Instituto de Ciências Biomédicas da UFRJ • Wilson Araújo da Silva Jr - Faculdade de Medicina de Ribeirão Preto/USP

Table of Contents

National Institute of Science and Technology in Stem Cell and Cell Therapy ............................................................................. 4

A) Program Description of the Institute ....................................... 4 The subject: Stem cells and Cell Therapy ................................ 5 Justification ..................................................................... 13

B) Objetives and Goals ........................................................... 14

C) Detailing the Research Program ............................................. 18 Lines of research and subprojects........................................... 39Studies with Pluripotential stem cells ...................................... 39Induced Pluripotent Stem Cells .............................................. 48Somatic Stem Cells ............................................................ 53Estabishment of Animal Models and Pre-clinical trials................... 74

INDICATORS ........................................................................ 86

D) Program for High Qualified Human Resources Formation ............. 87

E) Detailed Description of the Actions for the Transfer of Knowledge to Society ........................................................................... 95

F) Actions detailing for transfer of knowledge for the Business Sector or for the Public Policy formation ........................................... 101

G) Detailed Description of the Proposing Group ............................. 108

H) Activities Specification that must be done by the Team members .... 117

I) Mechanisms that will be used to promote the Interaction of the Research Groups ............................................................... 118

J) Forms of Interaction with Foreign Groups ................................. 119

K) Definition of the Specific Tasks of Each Entity ........................... 120

L) Comparative Analysis between the Current and Expected Statuses ... 121

M) Justified Budget ............................................................... 122

N) Potential for the Generation of Patents ................................... 145

O) List of the Projects Financed over the last 5 years ...................... 147

P) Formal Agreement by the Institutions Involved ........................... 153

Q) Institutional Contribution .................................................... 180

R) Time table ...................................................................... 193

S) Indication of Management Committée ...................................... 197

T) Organization and Functional Structure of the Institute ................. 198

References ......................................................................... 199

4

National Institute of Science and Technology in Stem Cell and Cell Therapy

A) Program Description of the Institute

The present proposal of creation a National Institute of Science and

Technology (NISCT) is to continue and to amplify the activities of Center for Cell-

based Therapy (CTC-CEPID-FAPESP), one Center of Excellence in Research,

Innovation and Diffusion created by FAPESP in 2001 with the aim objective to be a

new paradigm to organize research at Sao Paulo state. The CTC-CEPID, as we

expected to be NISCT in the future, develops activities in three sectors which

constitute the project scope: scientific project on the frontiers of knowledge,

diffusion of knowledge for society and technological innovation with close

collaboration with productive sector.

The best way to demonstrate our capacity to perform the proposed

objectives consists on demonstrating what we are doing at the present time. The

CTC-FAPESP is formed by eight principal investigators (PI) which had good scientific

research until 2000 but with very low interactions among of them. The impact of

creation of CTC is evaluated at figure 1. The academic production related to Cell

Therapy from the eight PIs increased gradually each year which resulted in 70

publications in 2007. The article citations of these PIs also increase significantly

and became higher than 600 citations in 2007. The score h of the group is, at the

present time, 42 which demonstrate the quality of scientific production. The

collaborations among of PIs, which is measured by the presence of co-authors at

the publications, also increased. All together, this demonstrates that CTC has

established a unique plan, promoting the convergence and integration of the

research areas among their PIs. Moreover, stimulate the researchers to actively

participate to the diffusion of scientific knowledge. As example, is the edition of

the book: “Stem Cell – the new frontier of Medicine” which became a reference in

this area in the country and received the Jabuti Premium in 2007.

The creation of a National Institute for Stem Cell and Cell Therapy (INCTC)

will allow for the consolidation and amplification of the experience obtained with

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CTC, and will allow the mobilization and aggregation with the best research groups

about stem cells and cell therapy in the country which will act as a network to

allow the best performance of each specific competence and guarantee an

accentuated advance capable to place Brazil in forefront of international scenario.

Figura 1. Evolution of number of papers and cited papers obtained from the eight

PI from CTC.

The subject: Stem cells and Cell Therapy

Cell therapy, by definition, consists on the use of cells with therapeutic

applications. These cells can be used in several ways: by intravenous injections to

have systemic actions or to protected organs or tissues such as bone marrow or

central nervous system (CNS); used locally or injected directly into the damage

tissue or organ to promote some regenerative effect or to protect.

The cells used on cell therapy strategies are different in relation to their

state of maturation or differentiation. They can be used as mature cells from

peripheral blood such as eritrocytes, leukocytes and platelets as occurs, for

example, in blood tranfusions, as well as, lymphocytes or dendritic cells which are

in vitro sensibilized to be used in the cell vaccine protocols; also, it can be used

stem cells or progenitor cells with the objective to repair or even total

reconstitution of damage organ or tissue. As example of the latter, we have the

consolidate example from more of 40 years of experience, the hematopoietic stem

cells transplantation derived by bone marrow or peripheral blood or umbilical cord

blood and which are able to reconstitute the total hematopoietic system.

6

Other examples of cell therapies well succeed with stem cells, either

embryonary or somatic stem cells in animal models and in the man, have emerged

in the last years and the search for new cell therapies more effective have

occupied, in a relevant form, the scientific literature.

The objective National Institute for Stem Cell and Cell Therapy (INCTC) is to

develop an extensive program with basic and clinic researchs to understand,

isolate, culture and with therapeutic application in animal models, as well as, in

humans stem cells from different sources: embryonary stem cells, somatic stem

animals and induced pluripotent stem cells. To better understand the mechanism

involved in the maintenance or re-initiation of pluripotency it will be studied

neoplasic stem cells derived from several tissues, in special stem cells from bone

marrow which give rises to leukemia and lymphoma.

In general, the scope of the scientific project is illustrated by figure 2.. The

stem cells which will be studied can be classified into two groups: pluripotent stem

cells and somatic stem cells. In the first group includes the embryonary stem cells

and the induced pluripotent stem cells. In the second group will include

hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), endothelial

progenitor cells and neoplasic stem cells (NSC). Additionally, it will be studied stem

cells poorly characterized, such as, epithelial cells from placenta and stem cells

from different regions of yolk sac.

These stem cells will be isolated, characterized morphologically and

functionally and cultured to become the source for several studies with focus on

understanding the functional properties and to identify genetic and epigenetic

molecular mechanisms involved in the induction and control of cell differentiation.

Several strategies and tools dominated by the participants will be used in these

studies, including: genomic, proteomic, cytogenetic, genetic, epigenetic,

immunology, embryology, cell biology and culture and the sistemic biology. These

cells also, will be cultured in large scale to be used in pre-clinical and clinical

protocols with animals and humans. Specifically, for the studies with animals the

Institute has the proposal of creation a Center for Pre-Clinical Studies and a Bank

of Stem Cells to allow studies in animal of several sizes (rat, histricomorfs, rabitts,

ovine, canine, equine, bovine and monkeys). This Center will be formed with the

participation of Faculty of Veterinary Medicine and Zootecnia of São Paulo – USP,

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Faculty of Zootecnia and Food Engineer in Pirassununga – USP, National Center of

Primatas from Institute Evandro Chagas from Belém-PA and Laboratory for animal

model studies from CTC. The infra-structure and scientific and technologic

competence is present in all of these Institutions and will allow the formation of a

network to perform pre-clinical and clinical studies without precedents in the

history of the country. Specially, the studies with primatas (monkeys from old and

new world) will add an important differential in the pre-clinical studies with stem

cells developed in Brazil. In humans, three clinical studies at phase I and II will be

conducted using mesenchymal stem cells: for diabets type I and to treat acute

graft versus host disease (aGVHD), developed by patients submitted to bone

marrow transplantation.

To execute this ambitious work plan a group of specialists and Institutions

with comproved scientific experience in several areas will put together: molecular

and cell biology, genetic, embryology, immunology, hematology, systemic biology,

chemistry of protein, veterinary and bioinformatics. The participation of these

scientists and their respective groups performed in an integrative and

complementary way. The proposal objectives and expected results are the result of

a multidisciplinary approach which focus on the achievement scientific objectives,

but at the same way integrative relative to more relevant and extensive questions.

We expected that this scientific multifaceted and multidisciplinary approach in this

specific subject will allow the fast grow of cell therapy program, consolidating and

amplifying the participation of Brazil in the international community and placing in

the first line of evidence concerning to this subject. The research activities are

described with detail at item C.

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Figure 2. Scope of Project

In parallel with scientific program we intend to develop intense activity

about scientific knowledge transfer for the society involving the public in general,

but also, and in special the professors from high school and their students. The

NIST intend to amplify the transfer activities which are made by the Cell-based

Therapy Center (CTC-CEPID-FAPESP), and will extend these activities to the other

Pluripotent Stem cells Somatic Stem cells

Genomics

EPC HSC Cancer SC

Embryonic Induced (iPS)

Placenta Epithelial cells Yolk Sac stem cells

Functional Properties and Mechanisms

Proteomics Cytogenetics Genetics Cell Biology Epigenetics

B. Systemic Immunology Embryology Culture Engineering

Diferenciation Controle and Induction

Production of Cell to Clinical Use

Embryonic Lineages Stablishment

Neoplasic Lineages Stablishment

Animal Models Humans

Mice Hystricomorphus Rabbits Sheep Suine Canine Equine Bovine Monkeys

DiabetesDECHA (1) DECHA (2)

MSC

Clinical and Pre-Clinical Studies

Neoplasic Mechanism and tranformation

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associated laboratories (São Paulo, Pirassununga, Rio de Janeiro e Belém) as well as

will perform new activities with greater abrangency.

The knowledge dissemination activities were always relevant in the CTC

program. In 2000, as part of the program it was constructed the “House of Science”

at the Hemocentro de Ribeirão Preto do HCFMRP-USP (Coordinating Institution of

the CTC). The “House of Science” is an area of 50 m2 which allocates 8

professionals with exclusive dedication to the knowledge diffusion and Science

teaching are allocated. This team is coordinated by Profa. Dra. Marisa Barbieri, a

retired professor from the University of São Paulo who has large experience in

teaching and training science or biology teachers. The construction of this place

was supported by several Institutions: Fundação Hemocentro de Ribeirão Preto,

FAPESP, Fundação Vitae and the University of São Paulo (scholarships linked to the

COSEAS program).

Among the principal activities developed we can mentioned: Courses of

specialization termed “The cells, the genome and you, professor” administrated

together with USA and the Education Secretary of São Paulo State. This course was

offer to more than 200 professor of High School from public and private schools The

university extension presential or distance courses of with duration of 30 hours; the

projects of scientific initiation from FAPESP; the courses of scientific initiation for

students of public schools termed “Talented students” and “Adopt a Scientist”,

being the first one a project with weekly meetings among senior investigators from

CTC, students and professoars and the second one, a project which consists to

develop simple research projects, liderated by graduated students from São Paulo

University with have the responsability to teach to a small group of students and to

generate educational material for science diffusion as the “Journal of Science”.

This Journal is at the 18a. eddition and is produced by students and distributed for

several schools of the region. The production of education games, theatrical play,

such as, “A cell’s agony”, confection brochures, such as, “Talking about HIV and

AIDS” which was financed by Ministry of Health and distributed to Schools of all

country, as well as, the publication of manuscripts about the CTC educational

project at Brazilian and international journals are other diffusion material

educational from the House of Science.

10

In 2004, the House of Science had the amplification of their activities with

the construction, supported by the University of São Paulo, which gave a House

from the University Campus of Ribeirão Preto, of MULEC -Museu e Laboratório de

Ensino de Ciências (from English - Museum and Laboratory for Science Teaching)

which contains a permanent exhibition with educational material produced during

the activities developed at House of Science. This new place receive visitations of

students and public in general. Other activities performed are: the participation

during the National Week of Science and Technology (2004, 2005, 2006, 2007), in

Scientific Conferences (SBPC), in programs of electronic media (radio and TV) and

in the impressa midia. All these activities and divulgation of science which are

promoved and supported by CTC are described with details at the site of House of

Science (http://www.hemocentro.fmrp.usp.br/projeto/casaciencia) and it will be

part of the educational program from INCTC. The figure 3 illustrate a summary of

the activities of knowlegded transfer from CTC.

Other educational activities and formation of human resources will include

summer courses offered annually and courses of professional formation. The CTC

offers annually, at January month, a summer course with destination to

undergraduate students from biology area from all the country. Until this moment,

it was offered 8 editions with the participations of more than 280 students. The

thematic of these courses have been: genomic, proteomic and stem cells. The

course comprehends two weeks and include several theoretical and fundamented

practical classes within this thematic. Moreover, CTC offers specialization courses

for physician and no physicians in the Field of hemotherapy, nurse, social

assistance and gestor in health service.

The CTC is linked to São Paulo University by the Faculty of Medicine of

Ribeirão Preto and other units situated at Ribeirao Preto Campus and their

professors and researchers which are activally engaged at undergraduated courses

of medicine, pharmacy, chemistry, biomedical informatic. Also, participate

regularly at pos-graduation courses from these departments.

11

2001 2002 2003 2004 2005 2006 2007 2008

“The cells, the genome and you Teacher” “Talent-Hunting”

“Site”“Journal of Sciences”

“Pre-congress” “Junior scientific

initiation”

“Curso On-Line Course ” “MuLEC” “National Week of Science and Technology” “Adopt a Scientist”

“Wheel of Science”

“Tuesday of Science”

“Wall I and II” “Fridays of

Science” “Wall land

II” “Folhetins” “PIPOC” “PIPAS” “Site to Teacher”

“Science for Everyone Space”

“Blood Panel”

“Congress of Pediatrics”

“Safety Blood”

“Partners on the Science Dissemination”

“Agony of a Cell”- Brasília

“Stem Cell Exposition”

“Sara and the Blood

preciousness”

“Pre scientific initiation - USP”

“Science with the youth – Building the future”

“Retro Room”

“Events and Activities”

Institutes of Support and Funding: FAPESP; CNPq; Fundação Vitae; Pró-Reitoria de Cultura e Extensão – USP

Figure 3. History of the Transfer Activities.

In this present proposal, the objective is to amplify the CTC experience and

to extend these described activities to the laboratories and units which are

associated in the INCTC proposal. Also, we plan to scale up these activities by

offering specialization courses via internet. The target public will be students and

science or biology teachers from High Schools. During the first year the INCTC has

planned to offer a specialization course of 360 hours to train 900 science or biology

teachers. As an integrant part of the activities, each professor will need to register

a classroom from a public school with at least 40 regular students. This initiative

will result in the participation of 36,000 students during a 10 month period. The

activities with the school teachers will be predominantly performed at distance

using the TIDIA plataform. Approximately 20% of the activities will be performed on

12

site at distance-education training places situated at different units from

universities of the State of São Paulo. The course will be authorized by the USP and

developed within the Program “Virtual University” of the State of São Paulo

(UNIVESP). The official documentation of the course is underway and a preliminary

approval from the Secretary of Higher Education of the State of São Paulo and from

the Pro-Rector of Culture and Educational Extension of the University of São Paulo

was already obtained. After the second year of the project, the course could be

extended to the entire country.

Moreover, we intend to organize an Inter-Units post-graduation course

(FMRP-USP, FMV-USP, FZ-USP, ICB-USP e UFRJ) on Stem cell and Cell Therapy and a

professionalizing master course for training specialized technicians in this field.

In addition to these activities we are proposing an ambitious plan of science

diffusion and specialized personnel training by offering post-graduation strictu e

latu senso courses. The divulgation of scientific relevant productions generated

within the INCTC will be performed by scientific publications and additional

supplementary means. The main goal is to reach several segments of the society in

order to promote the interest for science and demonstrate the importance of

continuous scientific education. These new initiatives proposed by the INCTC are

described with more details in the Item E.

Finally, the innovation activities, together with the research and education

transfer programs, constitute the triple helix that characterizes this project

proposal. The triple helix represents the ideal organization model of Universities

and Research Centers at the XXI century (Etzkowitz, 1996). The INCTC intends to

amplify the experience of the CTC-CEPID in implementing business incubators

within the Coordinating Institution. The first business incubator denominated

INBIOS (Business Incubator in Health Biotechnology) was successfully implemented

in 2002. The objective of INBIOS is to give support to business people to create or

amplify biotechnology business companies or to support special P&D areas of

middle and large companies interested in developing products or services at the

incubator company. The preferential working areas of INBIOS are: biotechnology,

biomedicine, medical and odontological materials and equipments, tools,

informatics technology, environment, chemistry and nuclear technicians. In 2006,

the INBIOS, was integrated to the SUPERA business incubator of Technology of

13

Ribeirão Preto, which was then denominated SUPERA-HEMOCENTRO

(http://www.fipase.org.br/default.asp).

At this moment, SUPERA gives support to 09 resident companies, 03 pre-

resident companies, 01 associated companies and 20 projects in the modality of

“Hotel of Projects”. The SUPERA-HEMOCENTRO is installed in the space of 128 m2

and offers space to companies, such as: Rad Tech Sistemas Médicos Ltda, Capelli

Fabris Desenvolvimento e Pesquisa Ltda, Lychnoflora Pesquisa e Desenvolvimento

de Produtos Naturais Ltda, CG Brasil Consultoria e Informática Ltda e Innolution

Sistemas de Informática Ltda.

The INCTC project will incorporate this previous innovation experience of

the CTC-CEPID into the productive sector. Additionally, it will be developed new

initiatives focusing in technology development and its transfer to the productive

sector, as detailed described in the Section F.

Justification

In the year 2005, the United Kingdom promoted a panel of specialists

diagnose and plan activities related to stem cells and cell therapy for 10 the next

years (2006-2015). In the final document, UK Stem Cell Initiative – Report and

Recommendations – short to long term actions were proposed to place the UK in

the leading position worldwide in cell therapy, which was considered strategic due

to its innovative potential in the healing of illnesses. In chapter 5 of the document,

a fluxogram describing the critical steps that would rule the development of

products and processes with stem cells and cell therapy was presented. In Figure 4

we reproduce that fluxogram. The development of new cellular therapies starts

with an effort in basic research in order to isolate, characterize and understand the

biological mechanisms involved in the maintenance of the undifferentiated status

of the stem cells. The second step in this fluxogram consists of the establishment

of well-characterized and homogeneous Stem Cell lines, which are to be deposited

in a stem cell bank specifically created for that purpose. The bank would be

responsible for storing, exporting and distributing the cells lines to research

laboratories. In UK that suggestion led to the creation of the UK Stem Cell Bank.

The third step consists in the large scale production of well-characterized stem

14

cells to be used in pre-clinical and clinical studies. In the UK, several initiatives

sponsored by both the government and by the private initiative have begun. At this

level, the generation of patents of products and processes is highly likely which

explains the great interest of the private sector in this area all over the world. In

the fourth step of the fluxogram, there are the clinical studies, which require

specialized physical and professional structures in that area. In the final stage,

there are the registration and launching of the product for general use.

In Brazil we can say for sure that most of the studies and initiatives are

found at phase 1 of the fluxogram below. The current proposal takes that track.

We propose a group of activities that are distributed throughout the described

phases described in Figure 4: basic research, the creation of a stem cell bank, the

development of large scale cultivation systems under GMP conditions, and pre-

clinical and clinical tests. This coordinated effort will put Brazil at the leading edge

of research in cell therapy.

THERAPIES PRODUCED FROM STEM CELL LINES

Basic Stem Cell Research

Stem Cell Bank Stem Cell Therapy

Production Unit

Stem Cell Clinical Trials

Licensed Product Manufacture

Cell Biology Research grade cell banking

and characterisation

Production process

development

Specialist Bed and Facilities

Stem Cell Therapy Imunology

Animal Modeling Safety and Efficacy

Commercial or Plubic Sponsor Clinical grade

cell banking and

characterisation

Plot scale capacity to

supply stem cell clinical trials

Epigenetics Coordination Therapy

Surveilance Bioengineering1,3,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20, 21.24 e 25 *

4,6,7,26, e 27* 2 e 23* 28, 29 e 30*

Figure 4. Fluxogram of the phases under development of cell therapies. (Adapted

from UK Stem Cell Initiative, 2005).* number of the subprojects.

Marketing Approval Cell Therapy Production

Stem Cell Clinical Research

Research & Development

Stem Cell Banking

15

In the last line of Figure 4 we show the relationship of the phases with

subprojects proposed here.

The major differential of this Project is that it expands the scope of the

stem cells studies and cell therapies that are currently underway in Brazil.

Although there are several phase I and II clinical studies underway in the country,

we note that these are generally based on the use of minimally manipulated bone

marrow mononuclear cells and follow experimental protocols established by other

studies, which became widespread worldwide. These protocols were embraced by

many opportunistic research groups which had no previous experience in studies

with stem cells simply because these cells are easily obtained. That phase was

rapidly exhausted, and it was demonstrated that the revolutionary clinical

advances expected from the use of the mononuclear fraction of bone marrow were

not realized, this reduce the priority of this kind of study in cell therapy. Cell

therapy, as we have previously defined, has great therapeutic potential, being

really able to revolutionize the treatment of countless chronic-degenerative

diseases. However, in order to reach such potential, both scientific and regulatory

activities are necessary. Unfortunately, this does not exist in a consolidated way in

Brazil at the present moment.

The Project of the National Institute for Stem Cells and Cell Therapy

proposes a model of coordinated performance, similar those adopted by the

countries who have become leaders in this area, especially the United Kingdom. As

shown in Figure 4, our proposal is constituted by a group of subprojects that aim to

accomplish both scientific and technical, so that effective cell therapy be available

for wider sectors of the population in a time span that ranges from 8 to 15 years.

In this context, a first group of subprojects is directed to the study the study

of the basic properties of stem cells (cellular biology, immunology, epigenetic,

genomics, cytogenomics, proteomics, bioengineering, etc.). Included in this group,

there is also the development of animal models, and here we emphasize the

utilization, for the first time in Brazil, of non-human primates for the development

of experimental models to test cellular therapies. We intend to form a Center of

Pre-Clinical Studies to meet the demand that will be generated by the project.

Simultaneously, a second group of subprojects will be developed who

objective is the establishment of homogeneous pluripotent and somatic stem cell

16

lines. Our goal is to obtain well-characterized stem cell lines that can be expanded

culture systems without losing their primitive properties. They will be made

available to several research laboratories and for pre-clinical and clinical studies.

To reach this goal, we intend to establish two stem cell banks: one for animal stem

cells, which will be coordinated by teams of Prof. Maria Angélica Miglino at Faculty

of Veterinary Medicine of the University of Sao Paulo (FMV-USP-SP) and by Dr.

Klena Sarges Marruaz da Silva at the Belem´s Primate National Center (Belem-PA).

The second bank for human stem cells which already exists at our Center for Cell-

based Therapy (CTC) - Hemocentro shall be enlarged in its processing capacity to

accomplish the needs required by the Institute. The major premise of this phase of

the Project is that the therapeutic effects obtained with the animal models and the

clinical studies need to be directly correlated with specific cell populations in

order to be reproducible and trustworthy and also be capable of expansion in

culture, so that the progress in this area can be scientifically consistent.

A third group of subprojects is directed to the development of large scale

stem cell production system to extend the availability of cellular therapies to a

greater number of patients. The limited expansion capacity of stem cells is today

the greatest obstacle to implement effective cell therapies. For instance, in the

clinical study of phase I we have been using mesenchymal stromal cells for the

treatment of the acute graft-versus-host disease (GVHD) post-transplant of bone

marrow, the treatment of one patient weighing 70 Kg demands the culture of cells

in 30 one liter culture flasks for 20 to 30 days to achieve a final number of about 7

x 107 cells. Without the development of more efficient cell culture system, it is

impossible to imagine that this kind of treatment may be extended to those who

need it. An alternative approach to circumvent this problem is to produce cells in

bioreactors. In the present Project, we will explore the use of bioreactors not only

for the expansion of embryonic stem cells but also for mesenchymal stem cells.

The fourth group of subprojects involves clinical studies using mesenchymal

stem cells to treat patients who have type 1 diabetes and for the treatment of

acute graft-versus-host disease (GVHD). Our group is the only in Brazil using

mesenchymal cells cultivated under GMP conditions for therapy.

In the proposal group, this Project of INCTC promotes great advances to the

country in studies involving stem cells and cell therapy.

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B) Objetives and Goals

To aggregate renomated Brazilian researchers involved in the study of stem

cells, currently working in different parts of the country, in a network called

Instituto Nacional de Ciencia, Tecnologia e Inovação (INCTI). The set up of this

INCTI will allow the interchange of scientific information, reagents, methods,

human resources and logistic, as well as equipment already available at the

different centers, in order to foster academic and technological advancement,

along with training of specialized personnel in the field of Biotechnology, Health

and Agrary sciences with national and international relevance.

One of our aims is to address essential questions such the characterization of

stem cells from different sources and species, determination of genetic, epigenetic

and cellular mechanisms regulating the maintenance of the pool and the

differentiation of stem cells. In addition, we propose to create technical protocols,

a tissue and cell bank, bioinformatics tools, cell lineages, genetically modified

animal which will be used in the subprojects listed below. The coordinated effort

of the different researchers and centers will be a unique opportunity to approach

the scientific problems using several strategies. As aims in applied science we

propose to use stem cells in the treatment of patients with diabetes mellitus,

multiple sclerosis and graft versus host disease.

Our proposal also aims to generate human resources in the field of

biotechnology, health and agrarian sciences. Moreover, we will create the learning

opportunities for teachers of biology and sciences working in middle and high

public schools, as well as arouse the interest of children and adolescents attending

these schools in the study of Sciences. The general population and technology

companies (some of them working in the SUPERA) will have access to the main

achievements of the Project.

To allow the monitoring of the development of the project, we list below

the specific objectives of each subproject (item C). This list, chronograms and

supplementary material may be found in specific sections and in the website

www.hemocentro.fmrp.usp.br/projeto/inct .

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C) Detailing the Research Program

The research program of the INCTC is composed by 30 subprojects which are

presented here with the objective and goals of each one. In the second part of this

item (C2) these subprojects are briefly described as part of a rational organization

indicating, preliminarily, the integrator character of the proposal. The

rationalization for this program was shown in figure 4. In the H item of this project

(Activities Specification that Must be Done by the Time Members) we present a

table which shows the integration of the involved research groups (represented by

the coordinator’s names) in these subprojects.

C1) Subprojects, Objectives and Goals

Subproject 1: “Evaluation of the epigenetic stability on embryonic stem cell

lines established under different conditions”

http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB01.pdf

Coordinator: Lygia da Veiga Pereira

1. The establishment of hESC lines from the inner cell mass of blastocyst;

2. Evaluation of different conditions for cell line establishment (in the presence

or not of murine fibroblasts; with fetal bovine serum or serum replacement;

in a defined medium) in relation to its epigenetic state;

3. Characterization of the plasticity of hESC lineages;

4. Analysis of the state of activity of X chromosomes in trophoblasts cells

removed from embryos;

5. Evaluation of the effect of chromosomal instability on capacity of hESC.

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Subproject 2: “Scale-up of human embryonic stem cell production in

bioreactors for application in regenerative medicine”


Coordinator: Stevens Kastrup Rehen

Specific goals are:

1. To evaluate comparatively the growth and pluripotency of human embryonic

stem cells cultured in stirred systems as embryoid bodies or using

microcarriers as a support.

2. To characterize chromosomal stability and differentiation properties of

human embryonic stem cells maintained in stirred systems for long periods

of time.

3. To scale-up the expansion of human embryonic stem cells by the use of

bioreactors with the objective to use them for transplantation in

experimental animals and, eventually, in humans.

4. To develop an economically viable protocol for the expansion of human

embryonic stem cells for the production of large amounts of cells.

Subproject 3: “Molecular basis of hematopoietic differentiation from embryonic

stem cells” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB03.pdf

Coordinator: Dimas Tadeu Covas

1. Standardization of an in vitro hematopoietic differentiation protocol for

human embryonic stem cell (hESC) lines;

5. Immunomagnetic selection of CD34+ hematopoietic stem cells (HSC) derived

from hESC and umbilical cord blood (UCB) and subsequent RNA isolation;

6. Standardization of in vitro T-lymphocyte differentiation protocol from CD34+

HSC derived from hESC or UCB;

7. Evaluation of differentiation protocols by flow cytometry;

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8. To perform microarrays to obtain the transcriptional profiles (mRNA and

microRNA);

9. Analysis of the results using bioinformatics and specific databases for

identification of putative regulatory mechanisms;

10. Validation of results by real-time PCR (mRNA and microRNA results).

Subproject 4: “Culture of adult and embryonic stem cells of animals and

human beings for use in cellular therapies”


Coordinator: Maria de Fátima Lima de Assis

1. Bibliographical survey for the choice of the protocol to be adopted for cell

culture;

2. Selection, collection and identification of the cells;

3. To culture adult and embryonic stem cells with minimum chemical

interference to conserve the genome integrity, keeping the cultures

cryopreserved in the first stage to prevent the occurrence of polyploidy in

vitro;

4. Culture, expansion and cryopreservation of embryonic and adult stem cells;

5. Karyotypic identification of adult and embryonic stem cells;

6. In vitro differentiation of the stem cells, into specific tissues, for purposes

of cell therapy;

7. Stimulate adult stem cells to develop function specific of embryonic stem

cells (cellular dedifferentiation).

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Subproject 5: “Gene Modification of stem cells”



1. To construct lentiviral vectors carrying the stemness genes followed by IRES

and GFP;

2. To transduce mesenchymal and endothelial cells with the viral vectors to

induce the enhancement of the transcription factors Oct3/4, Sox2 and

Nanog.

3. To evaluate the changes in the gene expression profile to elucidate the

mechanism of action of stemness genes and to describe new therapeutic

targets.

4. To select the mesenchymal cell clones transformed by the lentiviral vectors,

assess the viability and observe the capacity to differentiate into different

cell lines.

Subproject 6: “Establishment of induced pluripotent stem cell lines (iPS) in

large animal models” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB06.pdf


1. Development of pluripotency induction vectors in non-human primates and

canine cells;

2. Establishment of iPS lines from animal model fibroblasts;

3. Neural differentiation of iPS and pre-clinical assays in animal models of

spinal cord injury.

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Subproject 7: “Establishment of induced pluripotent stem cell lines (iPS) from

fibroblasts of patients with Mendelian and multifactorial genetic diseases (with

genetic component)” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB07.pdf


1. Standardization of methods for the generation of human iPS from skin

fibroblasts, peripheral blood and bone marrow cells;

2. Establishment of iPS lines from patients with genetic diseases of interest to

the groups, in order to study their molecular mechanisms;

3. To establish iPS from human fibroblast cell lines GM1662, 46 XX,

heterozigous for a HPRT gene mutation, in order to use this cell line to study

epigenetic modifications associated with human X chromosome inactivation;

4. To develop adenoviral bi-cistronic vectors aiming to (i) increase iPS

production efficiency and (ii) generate non-genetically modified through the

elimination of vectors after the induction.

Subproject 8: “Autologous pluripotent cells generated from differentiated

somatic cells” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB08.pdf

Coordinator: Flavio Vieira Meirelles

1. Dedifferentiation of differentiated cells using the following methods:

2. Genetic insertion of known genetic factors (iPS)

3. Nuclear transfer using fibroblasts as nuclear donor cells;

4. Nuclear transfer using iPS as nuclear donor cells;

5. Derivation of pluripotent cells with the aforementioned methods, as well as

from embryonic stem cells in vitro generated;

6. Morphological, gene expression and epigenetic comparison of

undifferentiated cells obtained using the methods above.

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7. To determine the method that most resembles the undifferentiated cells of

the control group (transcriptome, epigenomic and proteomic profiles) and

apply it in the interespecific model;

8. To perform dedifferentiation functional assays with the cells generated by

the interespecific model: bovine cytoplast and monkey cell as nuclear donor

cell.

Subproject 9: “Animal models to study mitochondrial inheritance intra- and

interspecies” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB09.pdf

Coordinator: Flavio Vieira Meirelles

1. To generate bovine blastocysts (Bos taurus) with mtDNA of somatic origin

intra- (B. indicus) or interspecies (Homo sapiens) in heteroplasmia with

mtDNA of embryonic origin (oocyte inherited)

2. To develop a method to increase the percentage of inherited somatic mtDNA

in the blastocysts (B. indicus or H. sapiens).

Subproject 10: “Basic knowledge of MSC biology by means of comparison with

pericytes” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB10.pdf


1. Compare freshly isolated pericytes, pericytes cultured under MSC conditions,

and MSCs in terms of their behavior when subjected to differentiation

conditions described for MSCs, and different culture conditions in vitro;

2. Compare freshly isolated pericytes, pericytes cultured under MSC conditions,

and MSCs in terms of their ability to contribute to diverse tissues when

injected into blastocysts;

3. Study the surface marker profile of freshly isolated pericytes, and compare

it directly to that of pericytes cultured under MSC conditions, and cultured

MSCs;

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4. Study the profile of cytokines with immunomodulatory potential expressed

by freshly isolated pericytes, and compare it directly to that of pericytes

cultured under MSC conditions, and cultured MSCs;

5. Observe the behavior of pericytes infused systemically in an animal model of

tissue injury, using cultured MSCs as a control, and investigate the role of

pericytes during the tissue repair/regeneration process.

Subproject 11: “Investigation of angiogenic process and molecular interactions

between mesenchymal stem cells or pericytes with endothelial cells using

developed in vitro and in vivo systems”



1. Phenotypic characterization of mesenchymal stem cells from bone marrow

(BM-MSC) and umbilical cord vein (UCV-MSC) and central nervous system

microvascular pericytes (CNS-P) using 6 CD markers: CD271, CD140B, 3G5,

NG2, Stro-1 and CD146. Among of them, to select the surface antigen which

is more expressed in MSC and CNS-P;

2. Clonogenic assays and morphological characterization of FACS-sorted BM-

MSC, UCV-MSC and CNS-P using a specific marker previously selected;

3. Morphological and phenotypical characterization of FACS-sorted cell

populations;

4. Isolation, morphologic and phenotypic characterization of mesenchymal

stem cells from canine yolk sac (cYS-MSC);

5. Investigation of multipotential potential of FACS sorted and expanded stem

cells using standard conditions for osteogenic, chondrogenic and adipogenic

differentiation;

6. Genetic modification of human and canine MSC with firefly luciferase (luc)

and and DsRed2 fluorescent protein using transposon-mediated gene

transfer;

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7. Morphological and phenotypical characterization of FACS-sorted cell

population and DsRed2 fluorescent protein positive cells;

8. Isolation and culture of human umbilical vein endothelial cell (HUVEC) and

canine yolk sac endothelial cells (cYS-EC) FACS-sorted according to CD31

high level expression;

9. Generation of mature endothelial cells from human bone marrow - and cord

blood–derived endothelial progenitor cells (CD133+KDR+);

10. Morphological and phenotypical characterization of FACS-sorted CD31+ cell

population;

11. The influence of endothelial soluble factors, as well as, the requirement of

direct cell-cell contact between endothelial cells and BM-MSC, UCV-MSC or

NS-P using co-culture in vitro systems;

12. Morphological, phenotypical and gene expression analysis of both cell

populations before and after co-culture systems, as well as, the formation of

vascular like structure on matrigel for the evaluation of in vitro

angiogenesis;

13. Assessment of the role of BM-MSC,UCV-MSC, CNS-P and cYS-MSC in

angiogenesis after co-infusion with HUVEC or cYS-EC using the Matrigel plug

assay followed by subcutaneous injection in NODSCID mice. Image of new

blood vessels in live animals with IVIS Xenogen system will be performed;

14. Characterization of new blood vessel formation by confocal microscopy;

15. Analyze the contribution of mesenchymal stem cells or pericyte together

with endothelial cells to the recovery of the hind limb ischemic injury in

NODSCID mice by confocal microscopy. .

26

Subproject 12: “Comparison of proteins Expression of human mesenchymal

stem cells (MSC) obtained from bone marrow and umbilical vein cord”


Coordinator: Lewis Joel Greene

1. To obtain human MSC from bone marrow and umbilical cord vein and

expansion of these cells in vitro.

2. Immunophenotypic characterization and MSC differentiation into adipocytes,

condrocytes and osteocytes.

3. Extraction and quantification of soluble cytoplasmatic proteins of MSC.

4. Bidimensional electrophoresis and comparison of the proteins in the MSC

extracts with fluorescent dyes (DIGE).

5. Gel 2D quantitative analysis using the DeCyder software.

6. Protein identification of the spots of interest obtained from preparative gel

after trypsin digestion by mass spectrometry (MALDI-TOF-TOF).

7. Proteins identifications using data banks such as the Gene Ontology.

8. Comparison the Proteome results to the published data obtained by SAGE

Subproject 13: “Differences in gene expression and immunophenotypic profile

of amniotic epithelial cells obtained with and without animal substances”


Coordinator: Wilson Araújo da Silva-Jr

1. - To obtain two amniotic epithelial cell populations using different methods

of isolation and culture, with and without serum;

2. - To evaluate typical markers of embryonic stem cells in these populations

by flow cytometry;

3. - To assess the expression of surface markers related to immunogenicity and

immunomodulatory properties of amniotic epithelial cells (flow cytometry);

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4. - Comparison of gene expression profiles of the different cell populations

obtained;

5. - To compare the differentiation potential into 3 germinal layers: endoderm

(hepatic); mesoderm (cardiomyogenic) and ectoderm (neurogenic).

Subproject 14: “Isolation and functional characterization of pluripotent stem

cell - side population”



1. Isolation and characterization of human side population of stem cells from

bone marrow, umbilical cord vein and blood capillaries from central nervous

system (day 0);

2. Isolation and characterization of human side population of stem cells

obtained from cultured mesenchymal stem cells (MSC-SP) isolated from bone

marrow, umbilical cord vein, as well as, cultured pericytes isolated from

central nervous system blood capillaries using the classical plastic adherence

protocol;

3. Isolation and characterization of ovine side population stem cells in bone

marrow and in blood capillaries from central nervous system (day 0);

4. Isolation and characterization of canine side population stem cells from yolk

sac (day 0);

5. Isolation and characterization of side population stem cells in bone marrow

and umbilical cord vein from transgenic fetal bovine, which express GFP (day

0);

6. Analysis of side population frequency from human, dog, sheep and bovine

tissues at day 0 by flow cytometry and in vitro clonogenic assay;

7. Analysis of side population frequency from human, dog, sheep and bovine

cultured mesenchymal stem cells tissues obtained by the classical plastic

adherence protocol;

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8. Culture and expansion of “side-population” of stem cells isolated at day 0,

as well as, SP cells isolated from MSC cultures using four different culture

media: a) culture medium for MSC expansion; b) culture medium for

expansion of hematopoietic stem cell (HSC-M); c) culture medium for

expansion of endothelial progenitor cells and d) culture medium for

expansion of human embryonic stem cells;

9. Analysis of karyotype and cytogenetic characteristics of human SP;

10. Analysis by microarray of the gene expression profile of human SP culture

and expanded under MSC conditions;

11. Culture and expansion of “side-population” stem cells isolated sheep, canine

and bovine tissues using medium from MSC expansion;

12. Morphologic and immunophenotypic characterization of side population stem

cells undergoing expansion using different conditions;

13. Differentiation potential of mesenchymal stem cells derived from human and

animal SP;

14. Analysis of the gene expression profile of human SP cultured under with

culture media (EPC, HSC and hES), as well as, bovine, canine and ovine SP

cells cultured with several media by real time PCR;

15. Investigation of therapeutic potential of human and animal SP using three

murine models: 1) long-term engraftment of hematopoietic stem cells after

bone marrow transplantation with human and animal SP expanded under HSC

medium conditions; b) engraftment of human and animal SP cultured and

expanded under EPC and MSC medium after endothelial hepatic injury by

monocrotaline administration and c) in vivo angiogenesis potential using the

Matrigel plug assay followed by subcutaneous injection in NODSCID mouse;

16. Investigation of therapeutic potential of culture SP cells using two large-

animal models: 1) ability of canine bone marrow SP cells cultured under HSC

conditions to stimulate hematopoietic reconstitution in a canine bone

marrow aplasia model and 2) ability of ovine brain capillaries SP cells

cultured under MSC and EPC conditions to engraft and persist in brain

ischemic injury.

29

Subproject 15: “Functional evaluation of microRNAs in mesenchymal stem cells

differentiation” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB15.pdf

Coordenador: Dimas Tadeu Covas

17. Isolation and expansion of bone marrow derived mesenchymal stem cells

from;

18. Differentiation under appropriate in vitro conditions into osteoblasts;

19. Quantification of microRNA expression during distinct stages of in vitro

osteoblasts induction;

20. Identification of potential gene targets for miRNAs by in silico analysis;

21. Functional evaluation of miRNAs that were differentially expressed.

Subproject 16: “Gene expression profile of mesenchymal stem cells of patients

with Osteogenesis Imperfecta during in vitro osteogenic differentiation.”


Coordinator: Wilson Araújo da Silva Junior

1. Analyze mesenchymal stem cells gene expression profile by microarray assay

at least in four points of osteogenic differentiation. These analyses will be

applied in two samples of patients with Osteogenesis Imperfecta and two

normal samples.

2. Select genes with differential expression to be evaluated by Real Time PCR.

3. To perform functional analyses of genes selected as potential regulators of

normal and abnormal osteogenesis evaluating cell proliferation, migration,

apoptosis and cell death and to verify if these processes are modified in

patients with Osteogenesis Imperfecta.

Subproject 17: “Genetic, proteomic and functional analyses of mesenchymal

stem cell (MSC) from patients with autoimmune diseases”

30


Coordinator: Julio Cesar Voltarelli

1. To isolate, expand and characterize (number of colony forming units-

fibroblast, immunophenotype, cell expansion, doubling time and

differentiation potential) MSC isolated from bone marrow of T1D and MS

patients;

2. To isolate, expand and characterize (number of colony forming units-

fibroblast, immunophenotype, cell expansion, doubling time and

differentiation potential) MSC isolated from bone marrow of T1D and MS

patients;

3. To evaluated the in vitro immunossupressive capacity of MSC from T1D and

MS patients

4. To evaluated the effect of the high dose immunossupression on the MSC from

T1D and MS patients;

5. To analyze the gene expression of MSC from T1D and MS patients by cDNA

microarrays;

6. To analyze the proteomic expression of MSC from T1D and MS patients by

two-dimensional gel electrophoresis;

7. To validate the gene expression results obtained by cDNA microarrays by real

time RT-PCR;

8. To study relevant genes/proteins, whose expression were found altered in

the gene and proteomic expression studies, by functional studies using RNA

interference.

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Subproject 18: “Determination of some proteins related to apoptotic process

and cell cycle control of hematopoietic and leukemic progenitors”



1. Isolation, separation, characterization and As2O3 treatment of normal

precursor cells, leukemic stem cell and leukemic blasts.

2. 2DE Gel standardization

3. Spots analyses by densitometry

4. Immune detection of proteins involved in the apoptotic process using mono

and polyclonal antibodies.

5. Protein identification by MALDI-TOF and ESI-MS/MS

6. Data analyze using bioinformatic approaches

Subproject 19: “Evaluation of the function of dyskerin in early hematopoietic

differentiation”


Coordinator: Eduardo Magalhães Rego

1. To examine the role of dyskerin in hematopoietic stem cells (HSC) of Dkc1

mutant mice will be tested against their wild-type counterparts in their

capacity for repopulating lethally irradiated recipients C57/BL6;

2. To examine the role of dyskerin in early lymphoid differentiation by

competitive repopulation assays using rag-/- mice, which do not have

mature T and B cells;

3. To quantify the expression of the Dkc1 gene in isolated progenitors by Real

Time PCR isolates by FACS Sorting in Dkc1m and wild type mice;

4. To analyze apoptosis and cell growth of Dkcm lymphoid progenitors and in

correlated cells in wild type animals.

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Subproject 20: “Acute Leukemia Model in Chlorocebus aethiops (Cercopithecus

aethiops) using haematopoetic stem cells transduced with retroviral vector

containing the hibrid gene CALM-AF10”


Coordinator: Eduardo Magalhães Rego

1. To establish the acute leukemia model in Chlorocebus aethiops using

haematopoetic stem cells transduced with retroviral vector containing the

hybrid gene CALM-AF10

2. To establish the collection protocol and bone marrow hematopoietic stem

cell culture

3. To study the haematology, cytogenetic, and molecular characteristics

4. Determination of bone marrow immunessuppression and transplantation

protocols

5. To establish the gene expression model by retroviral vector in bone marrow

haematopoetic stem cells from Chlorocebus aethiops

6. To study the haematological cytogenetic and molecular alterations due to

the CALM-AF-10 retroviral gene expression in the bone marrow of

Chlorocebus aethiops patients after the autologous transplantation.

7. To establish the leukemia treatment protocol in Chlorocebus aethiops.

Subproject 21: “Evaluation of the therapeutic effect of mesenchymal stem cells

on regeneration of extensive thermal burn wounds in animal model”


Coordinator: Júlio César Voltarelli

1. To compare therapeutic and regenerative potential of xenogenic and

allogenic MSC;

2. To compare therapeutic and regenerative potential of MSC administered

locally or systemically;

33

3. To compare therapeutic and regenerative potential of xenogenic MSC

isolated from bone marrow of wild- type C57BL/6 mice (Gal-1+/+) or Gal-1

deficient C57BL/6 mice (Gal-1-/-);

4. To evaluate the thermal burn wound severity and the skin regeneration

process by histological analyses of the wounded skin biopsies;

5. To evaluate the in situ expression of angiogenic and growth factors,

metalloproteinases, cytokines, chemokines in the wounded skin biopsies;

6. To analyze the differential protein expression of treated or non-treated

wounds by proteomic analyses.

Subproject 22: “Use of mesenchymal stem cells genetically modified with

human recombinant coagulation factor IX in Hemophilic B mice”



1. Isolation, expansion and characterization of murine hepatic mesenchymal

stem cells (mMSC);

2. Culture, expansion and characterization of human, hepatic mesenchymal

cell line termed LX2;

3. Gene modification of murine CTM (mMSC/FIX) and human MSC (hLX-2/FIX)

with human recombinant coagulation factor IX using retrovirus system;

4. Cell cloning of both recombinant cell lines to obtain a population with high

level of recombinant factor IX;

5. Characterization of both recombinant cell lines mMSC/FIX and hLX-2/FIX

about the expression level of rFIX by RT-PCR, as well as, the protein level

presents at the supernatant of cell cultures by ELISA;

6. Evaluation of biological activity of recombinant FIX present at the

supernatant of these cell lines by APTT assay;

34

7. Infusion of recombinant cells lines mMSC/FIX and hLX-2/FIX in NOD/SCID

mice and Hemophilic B mice;

8. Evaluation of engraftment level in the liver different times after the infusion

of each recombinant cell line;

9. Evaluation of FIX kinetics in the plasma mice different times after infusion of

each recombinant cell line;

10. Evaluation of the period of maintenance of FIX protein expression.

Subproject 23: “Bioprocess development for mesenchymal stem cell expansion

on microcarriers” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB23.pdf


1. Isolation, culture and expansion of bone marrow and umbilical cord

mesenchymal stem cells in static culture as control procedure;

2. MSC culture in spinner bioreactor on microcarriers

3. Comparison MSC cultured on microcarriers in spinner bioreactor and static

culture using five parameters: a) morphology; b) immunophenotypic profile;

c) cytogenetic profile; d) differentiation potential on adipocyte, osteocyte

and chrondocyte and e) analysis of gene expression;

4. Comparison the MSC culture performance in spinner bioreactor and static

culture related to: a) Population Doubling; b) Doubling time; c) cellular

viability and d) metabolic activity (glucose and glutamine consumption,

lactate and ammonium production among others);

Subproject 24: “Functional Proteomic: analyses of the nucleofosmine in the

gliomagenesis” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB24.pdf


1. To extend the obtaining of protein maps by bidimensional eletrophoresis of

the new patient samples.

35

2. Protein identification by mass spectrometry by MALDI-TOF-TOF

3. Introduction of the shotgun peptide sequencing proteomic strategy to obtain

additional the data from those of the bidimensional gels.

4. Proliferation, migration and apoptosis assays under EGF stimulation in T98G

and U87MG cell lines.

5. To obtain the bidimensional maps of protein extracts of T98G and U87MG

cell lines under EGF stimulation.

6. Identification of the differentially expressed proteins between the EGF

stimulated and non-stimulated cells.

7. Silencing or modulation of the nucleofosmine gene expression by

interference RNA (iRNA) in T98G and U87MG cells line with or without EGF

stimulation and functional studies (proliferation, migration and apoptosis

assays)

8. To obtain of the bidimensional maps T98G and U87MG cell lines proteic

extracts under EGF stimulation and silenced to NPM.

9. Study of the differentially expressed proteins (item “5”) in EGF stimulated

cells and silenced to NPM.

Subproject 25: “Cytogenomic tools applied to the investigation of chromossomic

instability in chronic lymphocytic leukemia (CLL)”


Coordinator: Roberto Passetto Falcão

1. Obtain metaphasic cells to use with the SKY technique

2. Hybridization of the metaphasic cells carry out to spectral analysis (Applied

Spectral Imaging)

3. Extraction of blood DNA from patients and healthy donors and the evaluation

of the change in the copy numbers of the genetic region by CGH array

method using the Agilent platform.

36

4. Identification and characterization of the breaking points related to the

presence of the chromossomic alterations; detection and identification of

the probably enrolled genes.

5. Identification of the submicroscopics deletions in patients with normal

kariotype.

Subprojects 26: “Center of pre-clinical studies and animal stem cell bank”


Coordinator: Maria Angélica Miglino

1. Identify, isolate and test culture protocols for olfactory neural epithelial cells

2. Develop methods for cellular differentiation and establishment of the ideal

markers for olfactory neural epithelial cells;

3. Isolation and characterization of mesenchymal stem cells of the amniotic fluid

and, yolk sac of canine fetuses to be applied in the treatment of genetic

"defects", in intrauterine environment.

4. Standardization of a protocol for genotyping using amniotic fluid and the yolk

sac of embryos/fetuses of dogs to detect the presence of genetic alterations

in prenatal period

5. Study the potential for differentiation in vitro of mesenchymal stem cells from

amniotic fluid and yolk sac of canine fetus, as well as, its potential for the

intrauterine injection and, study of biodistribution and differentiation into

multiple cell types

6. Application of the immature dental pulp cells in animal models of patients

suffering from total limbic deficiency and, demonstrating resistance to the

conventional surgical procedure.

7. 3-D reconstruction of critical defects in tibia of sheep and, characterization of

cell differentiation by immunohistochemistry.

8. Quantify osteoblasts and osteocytes in the selected 3-D complex after

application in vivo, using histological morphometry.

37

9. Establishment of primary culture of canine and feline osteosarcoma;

10. Characterization of tumor cells culture and analysis of proliferative potential

and cells markers;

11. Osteosarcoma in vitro regenerative therapy;

12. Establishment of human extravillus trophoblast cell;

13. Produce virus recombinants of retrovirus vectors derived from the Moloney

Murine Leukemia Virus (MoMLV) carrying the genes reporters LacZ and eGFP.

14. Transduce stem cells acquired from placenta using retrovirus vector carrying

the genes reporters LacZ and eGFP to analyze the expression of the genes in

vitro.

15. To evaluate the tracing of the transducing cells in mdx and nude mice, the

potentiality of differentiation and marking in the different tissues and, its

pathogenicity;

16. Trace the implanted stem cells in mice after euthanasia and histopathologic

analysis using the product of genes LacZ and eGFP.

17. Analyze the potentiality of immune modulated response against the human

extravillous trophoblast cells.

18. In collaboration to Prof. Dr. Dimas T. Covas (Dra. Kamilla Swiech) group,

knowledge of specific techniques for culture and expansion using microcarriers

will be acquired;

19. In collaboration to the researcher Aparecida Maria Fontes (member of Prof.

Dr. Dimas Tadeu Covas’ group) side population cells from of canine’s yolk sac

and liver will be established to test its the vasculogenesis potentiality.

Specifically to characterize the phenotype profile and genes expression using

specific markers. The responsibilities of the pre-clinical group will be to

establish the cerebral ischemic model and to establish in vitro the molecular

interactions of the relations with endothelium mesenchymal.

38

20. In collaboration with the researcher Dra.Lygia da Veiga Pereira, the group is

going to test chronic spinal lesions (canine model) the application of the iPS

(induced pluripotent stem cells) cells;

21. In collaboration with Dr. Lindolfo da Silva Meirelles, Dra. Irina Kerkis will

perform the injection of pericytes and mice Rosa26 mesenchymal stem cell

into blastocyst and, murines to evaluate the capacity of interaction in

different types of cells in embryos tissue.

22. In collaboration with Dra. Adriana Santos Moreno (member of Prof. Dimas T.

Covas’ group), we intend to inject GFP-neural cells from embryonic cells or

pluripotent stem cells into canine model with chronic medullar disease,

and/or in murine models with induced lesion, to verify the adhesion of these

cells in the injured tissue and, the development of the necessary synaptic

connections to the re-establishment of the functions;

23. In collaboration with the PhD student Carla Kaneto (member of Prof. Dr.

Wilson Araujo’s group), we intend to establish the mechanisms involved in the

imperfect osteogenesis that occurs during the differentiation process of the

osteoblasts. The results obtained from animal testing would identify the genes

involved in this process and, could be applied in human therapy.

Subproject 27: “To make Neotropical primates (new world) and Chlorocebus

aetiops (old world) to stem cell research as animal model for cellular therapies”


Coordinator: Klena Sarges Marruaz da Silva

1. Indentify good large animal models to stem cell research.

2. To establish clinical and pre-clinical model protocols of Neotropical primates

and Chlorocebus aethiops to test therapies using stem cells.

39

Subproject 28: “Treatment of Type 1 diabetes (DM-1) with mesenchymal stem

cell (MSCs)” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB28.pdf


1. To evaluate the safety (toxic effects, infectious and neoplasic complication,

mortality) of the intravenous infusion of the allogeneic MCS from related-

donor in recent-onset MD-1 patients;

2. To evaluate the therapeutic effect of MSC on the pancreatic autoimmune

injury of the MD-1 patients (determination of the levels of glycemia fasting ,

C-peptide and glycosylated hemoglobin before and after MSC infusions;

exogenous insulin needs before and after MSC infusions);

3. To evaluate the immune response (naive, memory and regulatory T cell

subsets, serum cytokine quantification, T cell repertoire, frequency of anti-

pancreatic β cells autoreactive T cells) in the patients in many periods after

MSC infusion;

4. To evaluate the immunological mechanisms (immunomodulation of the

pathogenic responses by the MSC; role of T regulatory cells) involved in the

therapeutic response of NOD mice (genetically determined diabetes model)

and C57BL/6 mice treated with streptozotocin (chemically-induced diabetes

model) to human MSC infusion;

5. To evaluate the regenerative mechanisms (role in the pancreatic tissue

regeneration; human MCS fusion with murine pancreatic β cells) involved in

the therapeutic response of NOD mice (genetically determined model) and

C57BL/6 mice treated with streptozotocin (chemically-induced diabetes

model) to human MSC infusion.

40

Subproject 29: “Treatment of multiple sclerosis with hematopoietic stem cells:

evaluation of clinical response and immune mechanisms of action”



1. To evaluate the safety (toxic effects, infectious complications, mortality) of

the HDI/AHSCT for treatment of relapsing-remitting MS patients;

2. To determine if the HDI/AHSCT could stop the disease progression in

relapsing-remitting multiple sclerosis patients, refractory to the

immunomodulatory action of Interferon;

3. To evaluate the efficacy of the HDI/AHSCT therapy in comparison to the

conventional treatment (Interferon, Copaxone or Mitoxantrone) for

treatment of relapsing-remitting MS patients;

4. To analyze the large scale gene expression by cDNA microarrays, of

peripheral blood CD4+ and CD8+ T cells from relapsing-remitting MS patients,

isolated at pre- and post-AHSCT;

5. To validate the gene expression results obtained by cDNA microarrays by real

time RT-PCR;

6. To study relevant genes/proteins, whose expression were found altered in

the gene and proteomic expression studies, by functional studies using RNA

interference.

Subproject 30: “Mesenchymal stem cell (CTM) for treatment and prevention of

graft versus host disease (GVHD) in transplanted patients with hematopoietic

stem cells” http://www.hemocentro.fmrp.usp.br/projeto/inct_eng/pdf/SUB30.pdf


1- Isolation and MSC expansion in large scale using culture standard medium

15% FBS;

2- Comparison of the MSC culture and expansion with human serum or platelets

lysate versus FBS;

41

3- Establish criteria for selection of donor MSC suitable for transplantation;

4- To determine the frequency of MSC using clonogenic assay to quantify the

number of colony forming units (CFU-F);

5- Characterization of immunophenotypic and cytogenetic profile,

differentiation capacity and immunomodulator potential;

6- Infusion of two MSC doses (1-2 x 106 CTM/Kg) in patients prone to develop

GVHD;

7- Infusion of 1 to 6 MSC doses (1-2 x 106 CTM/Kg) in acute GVHD patients non

responsive to steroids treatment;

8- Evaluate the early and late side effects of the MSCs infusion;

9- Analyze the engraftment time of the bone marrow;

10- To evaluate the incidence of the acute and chronic GVHD in patients which

received prophylactic MSCs

11- To evaluate the relapse of the hematologic disease after the transplantation

12- To analyze the immunologic recovery and leukocyte activation by flow

cytometry to T, B, NK lymphocytes and memory cells

13- Serum immunoglobulins quantification;

14- Evaluation of the acute and chronic GVHD development;

15- T cell and mesenchymal stem cell chimerism evaluation.

42

Lines of research and subprojects

Studies with Pluripotential stem cells

Pluripotential stem cells are capable of originating cells and tissues from the

three embryonic cell layers: ectoderm, mesoderm and endoderm. Because of this

characteristic, these cells are the most attractive from the therapeutic point of

view, once they would work in a wide range of diseases. Cells having this potential

can be obtained from the inner mass of blastocyst, fetal gonadal fold from 6 to 9

weeks old and from teratocarcinomas in adults. Recently, pluripotential stem cells

have been obtained from mature fibroblasts by the insertion of genes considered to

be inducers of pluripotentiality (Oct4, c-myc, Klf4 e Sox2), what gave birth to the

so called induced pluripotent stem cells (IPS). In the present proposal of INCTC one

of the focuses of study will be the pluripotential stem cells derived from the inner

cell mass of humans and animals and the IPS. For that, we are placing together four

groups of Brazilian researchers who have previous proven experience in this area.

The first group is headed by Dra. Lygia da Veiga Pereira, professor of Instituto de

Biociências da Universidade de São Paulo that has experience in cultivating

embryonic stem cells and other pluripotential stem cells, as the ones derived from

dental pulp (Sukoyan, Kerkis et al., 2002; Kerkis, Kerkis et al., 2006). The second

group is led by Stevens K. Rehen da Universidade Federal do Rio de Janeiro who has

great experience in the cultivation and neural differentiation of ESC (Mcconnell,

Kaushal et al., 2004; Kingsbury, Friedman et al., 2005; Rehen, Yung et al., 2005;

Rehen, Kingsbury et al., 2006). To this initial nucleus, there is the incorporation of

the group from Faculdade de Zootecnia de Pirassununga da Universidade de São

Paulo, which has great experience in the derivation of embryonic stem cells, in

embryology, and in manipulation techniques of oocytes, including the transfer of

somatic nucleus and the manipulation of mitochondrial content of the zygote

(Meirelles e Smith, 1998; Meirelles, Bordignon et al., 2001; Meirelles, Caetano et

al., 2004; Ripamonte, Merighe et al., 2005; Ferreira, Meirelles et al., 2007;

Miglino, Pereira et al., 2007; Biase, Fonseca Merighe et al., 2008; Paneto, Ferraz et

al., 2008). So far, in addition to the experience of the groups cited above, there is

also the experience of the group from Ribeirão Preto regarding cultivation,

characterization and expansion of stem cells under conditions of good

manufacturing practices (GMP) e in intrexpansion of stem cells in cellular lineages

(Covas, Siufi et al., 2003; Silva, Covas et al., 2003; Panepucci, Siufi et al., 2004;

43

Covas, Piccinato et al., 2005; Pereira, Faca et al., 2005; Carrara, Orellana et al.,

2007; Picanco, Heinz et al., 2007; Covas, Panepucci et al., 2008; Picanco-Castro,

Fontes et al., 2008; Picanco-Castro, Russo-Carbolante et al., 2008).

This group of laboratories and researchers have complementary experiences

and competences, what will allow the consolidation of a strong national group

dedicated to the study of embryonic and induced stem cells (IPS) and, certainly,

will place Brazil among the countries in the world that are in the vanguard of this

field.

After what has been discussed, the group coordinated by Dra. Lygia da Veiga

Pereira of USP (USP-SP) through subproject 1, which is entitled “Evaluation of the

epigenetic stability on embryonic stem cell lines established under different

conditions”, will have as general goal, to evaluate the epigenetic state of new

lines of human ESC, to establish conditions and characterize their plasticity in vitro

and in vivo. In addition, the trophoblast cells removed from the blastocyst during

the isolation will be used to study the beginning of the inactivation process of X

chromosome. For that matter, the following technical approaches will be used:

Micromanipulation of human blastocysts for isolation of ICM and trophoblasts;

cultivation of ICM; immunofluorescence and FACS studies of pluripotent cell

markers (OCT4, NANOG, SSEA-1,2,3,4); differentiation embryo bodies and

characterization by immunofluorescence; formation of teratomas in rats SCID;

analysis of the activity state of chromosome x in CTEh 46,XX pre and post

differentiation; FISH for detection of RNA of XIST; ChIP for modified histons; allele-

specific expression of X-linked genes; global methylation analysis of hESC by

methylation microscope.

Pre-clinical studies and the perspective of cell therapy in human require

quantities of cultivated stem cells in vitro. Therefore, defining good conditions for

cultivation, both in a small scale or large scale, is essential to the feasibility of this

and other procedures to be developed.

Few advances have been reported relative to the propagation of embryonic

stem cells in large scale (Thomson, 2007). The optimization of cultivation

conditions is usually performed in static dishes, that is, in low scale. However, such

procedure needs serial passages, which makes difficult the adequate control of

cellular physiology, driving the cells to frequently suffer; and also


44

manipulator/researcher interference which increases the risks of contamination

and makes expansion a hard process. For those reasons, it is essential to develop

new methods of expansion for human embryonic stem cells that will allow an

adequate control of environmental conditions, in a way that large scale cultivation

preserves the phenotypic characteristics, keeping the human embryonic cells as

pluripotent and homogeneous population. Cultivation in agitated systems seems to

be in particular most promising once they provide a homogeneous environment, in

which conditions such as pH, oxygen content and temperature can be constantly

monitored and controlled, allowing better control of the cellular physiology.

Furthermore, an agitated system eliminates the heterogeneity of the dish

environment, a static surface of cultivation. Due to its well known dependence in

cell-to-cell adhesion and formation of aggregates for proliferation, there are two

alternatives for the cultivation of embryonic stem cells in agitated systems: the

cultivation in suspension as aggregates and embryonic bodies, or the cultivation

when adhering to microcarriers. Microcarriers are porous particles, usually

spherical, showing high surface area and low density, which keeps itself in

suspension under soft agitating conditions, acting like a cell adhesion support.

Analysis of viability of human embryonic stem cells cultivated in agitated system,

comparing different matrices for microcarriers and the influence of different

substrates in large scale expansion, becomes extremely interesting.

To this end, the group coordinated by Dr. Stevens Kastrup Rehen at

Universidade Federal do Rio de Janeiro proposes subproject 2, entitled “Scale-up

of human embryonic stem cell production in bioreactors for application in

Regenerative Medicine”, which has the large-scale production of human

embryonic stem cells for use in preclinical and clinical trials as its general

objective. To achieve this goal, human embryonic stem cells obtained as a gift

from the Scripps Research Institute – USA, and from Harvard University (Cowan et

al. 2004; Klimanskaya et al., 2004) will be initially cultured over a monolayer of

mouse embryonic fibroblasts (MEFs; feeder layer), and submitted to different

expansion methods that will be compared to each other. Stirred culture systems

will be used to expand these stem cells as embryoid bodies in suspension as well as

in the presence of commercially available microcarriers (Cytodex 1, Cytodex 3, and

Cytopore – GE Healthcare). Conical flasks (erlenmeyers) and spinner flasks (Bellco)

will be used in stirred culture assays. For assays using automated stirred


45

bioreactors, a Bioflo 110 bioreactor system (New Brunswick Scientific) will be used,

and its native vessel, which has a standard geometry (0.4 to 1 L working volume),

will be compared with a vessel especially designed for stem cell culture (DASGIP,

0.1 L working volume). Operational variances of the process, e.g., stirring, aeration

rate and operation mode (batch, feed-batch and perfusion) will be evaluated. Cell

growth kinetics will be evaluated and molecular and immunocytochemical assays

will be performed with the purpose of evaluating and quantifying the cells’

pluripotency and degree of differentiation. The total cell concentration of the

cultures will be determined by counting cell nuclei in a Neubauer chamber under

the microscopy (crystal violet method). Glucose and lactate concentrations in the

supernatant will be determined using an YSI model 2700 automatic analyzer.

Chromosomal stability and other pluripotency parameters of ESCs cultured in

bioreactors will be compared with those of ESCs cultured under standard conditions

(MEFs; feeder layers) using newly developed protocols and methodologies for the

analysis of morphology and karyotyping (Martin, Muotri et al., 2005). Chromosomes

stained with 4’,6-diamino-2-phenylindole (DAPI) will be counted according to the

methodology developed by the team (Rehen, Mcconnell et al., 2001), in a minimum

of 80 chromosomal spreads. Molecular karyotyping and fluorescent in situ

hybridization (FISH) probes will be used if required (Rehen, Yung et al., 2005).

Alterations in proliferation, differentiation and survival of ESCs maintained in

culture dishes or bioreactors will be examined by immunohistochemical assays for

the bromodeoxyuridine (BrdU), phospho H3 (mitosis), Tuj-1, caspase-3, Ki67 and

DNA fragmentation, as previously described (Rehen, Varella et al., 1996; Rehen,

Neves et al., 1999; Rehen, Mcconnel et al., 2001; Rehen, Kingsbury et al., 2002,

Kingsbury, Rehen et al., 2003).

Despite the establishment, propagation and characterization of the cell lines

are a fundamental step in the eventual establishment of treatment protocols, to

the safety use of hESC it’s necessary that efficient and practical approaches be

developed to promote the desired differentiation. The success in this task could

avoid the appearance of unwanted cells or tissues, with uncontrolled growth or

other unexpected behave. Thus, the study of different processes of differentiation

is crucial for the possible implementation of ESC in clinical practice.

Different adult tissues have the capacity for renewal and repair, such as

blood, epidermis and liver tissue. The ability to repair or to renew some adult

46

tissues is related to the presence of adult stem cells that have a limited potential

for differentiation. Among these cells, the hematopoietic stem cells (HSC) are

highlighted because of the volume of theoretical and applied research in medicine.

The HSC are used for over 30 years as a form of hematopoietic reconstitution

treatment through the bone marrow (BM) transplantation. Similarly, newborns

blood that remains in the umbilical cord (UCB) and placenta after birth can be used

as a source of CTH. The success of the transplant depends, among other factors, on

the existence and compatibility between donor and recipient, since incompatibility

in this case may result in the disease graft versus host disease (GVHD). However,

the available cell number in the cord blood represents a limit that results in further

delay in restoring the hematopoietic system, increasing the infection risk in the

patient. Front to these restrictions, the production of HSC from ESC represents an

attractive alternative, which would eliminate this kind of restrictions.

However, HSC derived from ESC have differences concerning to the

engraftment potential (Shojaei e Menendez, 2008), which reflect the deficiencies

in homing, repopulation in long periods, and also in the differentiation potential,

specially the lymphocytic line (Martin, Woll et al., 2008). The comparison between

the gene expression profile of HSC derived from the BM or UCB have been

successively done by our group to characterize the molecular differences related to

the biological differences among these cells (Panepucci, Calado et al., 2007).

Similarly, the comparison among the adults HSC and ESC expression profile could

help in understanding the molecular base related to these existing differences, and

in the differentiation process optimization (Martin e Kaufman, 2005)

The group coordinated by Dr. Dimas Tadeu Covas of USP in Ribeirão Preto

(USP) through subproject 3 entitled “Molecular basis of hematopoietic

differentiation from embryonic stem cells”, will have as general goal, the study

of transcriptional profile of the ESC and of the HSC derived from those, comparing

them to the profile previously obtained from adult HSC. In addition to that, the

differentiation of the HSC (derived from ESC or adults) in T lymphocyte will be also

evaluated. This way, molecular differences identified can indicate which processes

of signaling are altered and their linkage to the deficiencies presented by HSC

derived from ESC. With this objective, commercial lines of ESC (or derived in other

projects) will be differentiated in HSC, using co-culture system with OP9 stromal

cells. The HSC will then be selected by immunomagnetic techniques using anti-


47

CD34 antibodies. Populations of HSC CD34+ of UCB will be obtained by the same

way. These cells (ESC and HSC) will have the transcriptional profile acquired by

using microarrays of oligonucleotides covering the whole human genome. The same

RNA of these cells will be used to obtain of the profile of the expression of

microRNAs. Additionally, HSC derived from ESC or UCB will be submitted to a

differentiation process in T lymphocyte, through the co-culture with OP9-DL1 cells.

These cells, the agonist ligand of the NOTCH (Delta-like 1) pathway induced to

differentiation Co-cultured cells will also be evaluated in terms of mRNA e

microRNA using microarrays. Bioinformatics methods and specific databases will be

used to integrate the information obtained so that it will identify potential

regulatory mechanisms involved in existing functional differences. Specific

antibodies will be used to evaluate the differentiation processes through flow

cytometry. Real Time PCR will be used afterwards to validate these differences

found in the mRNA as well as in microRNA.

The future of the cell therapy with stem cells will depend on the diffusion

and expansion of the area as a whole. This expansion depends on the establishment

of new centers of research, experienced on propagating the knowledge and the

techniques used in the study of the stem cells, forming specialized personal in this

area. With this in mind, the center intends to support the consolidation of the

group headquartered in the Instituto Nacional de Primatas Evandro Chagas in Belém

(Pará), as an operating group in the research with stem cells. The Seção of Meio

Ambiente of the Instituto Evandro Chagas makes use of a bank of cultivated

primary cells from the biopsy of humans and of other mammals, belonging to

Brazilian Amazon region. The bank is composed of cultures of some tissue and

organs such as: skin, kidney, epiphyses, brain, cornea, spleen and parotid gland.

The bank contains human cells of prepuce, cornea, sclera, quotes to umbilical and

placenta, beyond gotten stem cells from deciduous tooth (Miura et al., 2003).

This previous experience in the cell culture of distinct animal tissues,

successfully allows the training and implantation of the techniques for ESC culture.

In such a way, the Laboratory of Cell Culture of the Seção de Meio

Ambiente/IEC/SVS/MS of the Instituto Evandro Chagas kept for Maria de Fátima

Lima de Assis, through subproject 4, entitled “Culture of animals and humans

adult and embryonic stem cells for use in cell therapies”, it will have as general

objective to establish lineage of ESCs of animals and to characterize them.


48

Induced Pluripotent Stem Cell

When eventually mastered, the processes involved in the obtaining,

cultivating and differencing ESC into cells of clinical interest, another practical

limitation should be taken into account. In spite of the fact that HSC Banks may

eventually be created allowing the establishment of large collections, the ESC

would have their use restricted to hystocompatible receptor. One of the

alternatives to the creation of an ESC bank involves the reprogramming of the

nucleus of somatic cells in the case of a patient with no donor, by the cytoplasm of

an oocyte to obtain autologous hESC. This approach is also known as therapeutic

cloning, since it does not want to generate a cloned individual, but only ESC for

therapeutic use.

The reprogramming of the somatic nucleus induced by oocyte cytoplasm

resulted from the presence of different molecules, including transcription factors

and other proteins. Recently, several groups reported the induction of pluripotency

in human primary fibroblasts through their transduction with viral vectors

expressing the OCT4, C-MYC, KLF4 and SOX2 genes. The so called induced

pluripotential stem cells (iPS) have the characteristic morphology of ESCs. Express

pluripotential cell markers and are capable of differentiation in vitro and in tissues

derived from the three embryonic layers. This way, the generation of iPS from

somatic cell induction by specific factors could represent an alternative to obtain

of hystocompatible stem cells.

Preliminary results of Dr. Dimas Tadeu Covas group from Hemocentro de

Ribeirão Preto (USP) show the efficiency in the lentiviral production, in the stem

cell transduction (mesenchymal and endothelial progenitor) and also in the

endothelial progenitor expression profile with Nanog. Thus, this group, with the

subproject 5, named “Gene Modification of stem cell”, has as general objective

to genetically modify mesenchymal stem cell and endothelial progenitor cells with

lentiviral vectors carrying the stemness genes, in order to transform multipotent

cells into pluripotent ones with greater in vitro expansion capacity. The project

has the following approaches: a) To transduce MSC with 1054-CIGWS lentiviral

vector carrying one of the transcription factors (Nanog, Oct3/4, Sox 2, β-catenina,

Kfl4, c-myc, Esrrb, Tcl 1and Tbx 3) together with the GFP gene sort out the positive

cells by flow cytometry; b) transductions will be made one at a time and


49

afterwards four vectors at the same time will be used to induce de iPS; c)

Evaluation of the changes in the gene expression profile of the transduced cells; d)

Evaluation of the morphological changes of the transformed cells; e) assess the

specific embryonic stem cells markers (alkaline phosphatase and/or SSEA-1

antigens) e) evaluate if the modified cells have acquired pluripotenciality.

A great limitation of pre-clinical studies with ESCs is the lack of large animal

models on which these test can be performed since a series of technical/ biological

difficulties prevent the establishment of ESCs from embryos of those models.

Keeping this in mind, the group coordinated by Dr. Lygia da Veiga Pereira of USP

(USP-SP) through subproject 6, entitled “Establishment of induced pluripotent

stem cell lines (iPS) in large animal models”, intends to develop a methodology to

establish lines of iPS from large animal models specifically from non-human

primates and canine. The cells generated will be used for pre-clinical studies of

lesion of spinal cord in the respective animal models. For that matter, lentiviral

vectors of AddGene enterprise (EUA) will be transferred to 293 cells by co-

transfection with packing vectors. Cultures of dogs and monkeys will be transduced

with lentivirus expressing the reporter gene GFP for evaluation of transduction

efficiency with different packing systems; fibroblast transduction animals with

induction vectors according to conditions established above, and isolation of iPS in

culture medium specific for ESC (in case we are not able to establish the iPS by

using vectors with human genes of induction, the homologous of each species will

be isolated by RT-PCR from embryos, pre-implantation and new species-specific

vectors will be constructed and used for induction); characterization of

pluripotentiality of iPS by immunofluorescence, in vitro differentiation in

embryonic bodies and in vivo by the measurement of the formation of teratomas;

neural differentiation of animal iPS by culture in media with retinoic acid;

transplant of differentiated cells in spine cord lesion models.

Despite the fact that is inadequate for clinical use, the iPS are an important

tool of basic research, mainly for cells obtained from individuals with genetic

diseases Thus, the group headed by Dr. Lygia da Veiga Pereira , through subproject

7, entitled “Establishment of induced pluripotent stem cell lines (iPS) from

fibroblasts of patients with Mendelian and multifactorial genetic diseases (with

genetic component)”, intends to implement the methodology of generation of

human iPS so that it is possible to establish iPS from different tissues of patients




50

with genetic diseases of interest to the group, particularly patients who have

osseous dysplasia, type 1 diabetes, multiple sclerosis and acute promyielocytic

leukemia. The iPS established will be used as experimental model for the study of

basic mechanisms behind the respective diseases. Furthermore, new vectors based

on adenovirus will be constructed, which will not integrate to the genome, so non-

modified iPS will be generated, which is better suited for clinical use. For that

purpose, the following experimental approaches will be used: lentiviral vectors of

AddGene enterprise (EUA) will be inserted into 293 cells by transient co-

transfection with vectors; frozen mesenchymal cells of the bone marrow of

patients who have type 1 diabetes and leukemia PMA cells will be expanded in

culture for viral transduction; standardization of line establishment of adequate

cells for induction from human peripheral blood; transduction of human cells with

lentiviral vectors and selection of iPS cells in culture medium for ESCs; post-

immunofluorescence characterization of iPS and FACS using pluripotent cell

markers (OCT4, NANOG, SSEA-1,2,3,4); differentiation into embryoid bodies and

characterization by immunofluorescence; teratoma formation in SCID mice;

characterization of pluripotency of iPS through immunofluorescence, perform

differentiation into embryoid bodies in vitro, and teratoma formation assays in

vivo.

Although techniques for the induction of pluripotency involving the

incorporation of transcription factors in the genome of somatic cells (Takahashi et

al., 2006) constitute a possible approach to autologous cell therapy, it is important

to stress that the practical application of cells induced to differentiation by genetic

modification still has to be studied further and evaluated (Liu, 2008). On the other

hand, the technique of nuclear transfer (NT) is well-established and has been

capable of producing healthy animals, confirming the efficient capacity of

reprogramming of a differentiated cell (Wilmut et al., 1997; Keefer et al., 2000).

Additionally, when used as a receptor, the cytoplastic bovine cell has been capable

of reprogramming differentiated cells from various species (Chen et al., 2002,

Ilmensee et al., 2006), supporting the initial view that embryonary development is

necessary for the isolation of embryonary stem cells. Together with the efficient

reprogramming of the cytoplasm, the abundance and the ease in obtaining

biological material makes the bovine the perfect model for the establishment of

efficient methods for obtaining pluripotent cells of embryonic origin through the

51

reprogramming of differentiated somatic cells of various species (Cibelli et al,

1998).

By keeping that in sight, the group coordinated by Dr. Flavio Vieira Meirelles

(from now on called group) of USP in Pirassununga (USP) through subproject 8,

entitled “Autologous pluripotent cells generated from differentiated somatic

cells”, will have as a general goal to characterize methodologies to obtain

pluri/totipotent cells from somatic cells, in term of morphology, genetic expression

and epigenetic studies. For that matter, a bovine somatic cell line derived from the

in vitro cultivation of adult fibroblasts will be initially established. Part of these

cells will be used in the lentiviral transduction of transcription factors responsible

for cellular dedifferentiation (Takahashi et al., 2006). Such cells will be

characterized in the bovine model and, along with the non-modified cells; they will

be used as nuclei donors in the process of nuclear transfer. Embryonary cells

derived from those processes will be compared among themselves and with those

somatic undifferentiated cells in terms of morphology as well as genetic expression

and epigenetic expression. They will also be compared to the embryonary cells

obtained by the natural fertilization processes. The one methodology that provides

characteristics closes to the ones of the control group will be considered the most

appropriate for the production of stem cells from differentiated somatic cells and,

so, it will be utilized in the interspecific model where pluripotency cells of

primates will be produced.

The reprogramming of the nucleus of somatic cells by the cytoplasm of an

oocyte implies in the combination between the nuclear genome of an individual

and the mitochondrial of another. Considering the great potential of this kind of

approach in the development of the future of cell therapy, the control of the

inherited mitochondrial is very important to make possible the following steps: i)

the production of human stem cells by the reprogramming of the nucleus donor cell

in non-human cytoplasm and, ii) the production of autologous human stem cells

(Illmensse et al., 2006). For that matter, the bovine model is interesting because of

the abundance of material available and for the great quantity of available

knowledge on the mechanisms that regulate the mitochondrial DNA (mtDNA) during

embryogenesis. In bovine embryos produced by nucleus transfer of somatic cells,

the percentage of mtDNA of the nucleus donor cell rises between the third and

fourth cellular cycle in relation to the mtDNA that comes from the oocyte (Ferreira


52

et al., 2007). On the other hand, the centrifugation of bovine zygotes makes the

mechanical depletion of part of the mitochondria possible without compromising

the embryonic development because the embryo is capable of replacing the same

content of mtDNA observed in undepleted blastocysts (Chiaratti et al., 2008).

Furthermore, through mitochondrial depletion it is possible to introduce a greater

quantity of exogenous mitochondrial zygotes (Ferreira et al., submitted).

After what has been exposed, the group coordinated by Dr. Flavio Vieira

Meirelles (from now on called group) of USP in Pirassununga (USP) through

subproject 9, entitled “Animal models to study mitochondrial inheritance intra-

and interspecies”, will have as a general goal, to evaluate the viability of

production of embryos having mtDNA of inner and interspecific somatic origin and

to increase the percentage of such mtDNA inherited by the blastocysts. More

specifically, they intent to produce bovine blastocysts (Bos taurus) that present

mtDNA from inner or interspecific somatic origin (B. indicus or Homo sapiens,

respectively), in heteroplasmia with mtDNA from embryonic origin (inherited from

the oocyte) and; additionally develop a method to raise the inherited percentage in

blastocysts of somatic mtDNA (B. indicus or H. sapiens). For that, the following

technological approaches will be used – the establishment of mesenchymal cell

lines originated from the B. indicus and H. sapiens; enucleation of mesenchymal

cells by centrifugation and cytoplasm fusion for utilization as a cytoplasm donor

(Marchington et al., 1999; Shay et al., 1975); in vitro production of bovine zygotes

(B. taurus) parthenogenetic oocytes aspirated from ovaries collected on the

slaughterhouse and matured in vitro (Méo et al., 2007); centrifugation of zygote to

concentrate the mitochondria in one of the poles of the embryo and the removal of

part of the mitochondria through micromanipulation (Ferreira et al., submitted);

cytoplasts fusion (B. indicus or H. sapiens) to deplete zygote (B. taurus) and in

vitro cultivation (Inoue et al., 2000); determination of the percentage of mtDNA

(somatic mtDNA in relation to the total quantity of mtDNA) by PCR in real time

immediately after fusion, at 72 hours (embryos with 5 or more cells) and at 168

hours (blastocysts) after parthenogenic activation (Ferreira et al. submitted;

Ferreira et al., 2007). The percentage of mtDNA will be analyzed considering the

following as effect - the cytoplasm used (B. indicus and H. sapiens), the moment of

the analysis (0, 72 and 168 hours) and the interaction both factors.


53

Somatic stem cells

In the adult individual, different tissues have the capacity of renewal or, at

least of partial or total repair, the blood and the epidermis for instance, are

constantly renewing themselves, while other tissues, such as the liver, can be

repaired totally. Tissues, like muscles or neural present a reduced potential of

repair. Such potential is related to the existence of stem cells that are able of

proliferating and differentiating into different cellular types, the so called adult

stem cells.

Among those, we can highlight the hematopoietic stem cells (HSCs) and the

mesenchymal stem cells (MSCs). Both can be found in the bone marrow (BM). The

MSC differentiate in vitro and in vivo into the four main cellular types that form

the micro-architecture of the bone marrow: adipocyte, chondrocyte, osteocyto and

stromal cells. The interaction of the HSCs with this microenvironment allows the

constant renewal of blood tissue.

Opposed to the ESC, therapies based on HSC have been used for over 30

years, in the reconstruction of the hematopoietic system through bone marrow

transplantation. In the transplant, the incompatibility between donor and receptor

can result in graft-versus-host disease, or GVHD (graft versus host disease), when

the transplanted cells recognize the receptor as “nonself”. The mesenchymal stem

cells can be used in the reduction of GVHD in transplants, once there is strong

evidence that they reduce the immune response for their effect and T and

dendritic cells.

In addition to their use in reconstructing hematopoietic system,

mesenchymal and hematopoietic stem cells have been used to repair or replace

damage or sick tissues. These approaches are based on the observation that those

cells, originated in the mesoderm, could differentiate themselves (or

transdifferentiate) into cells of tissues originated from other embryonic layers,

such as hepatocyte (endoderm) or neuron (ectoderm) a process called plasticity. In

fact, these cells have been used in the attempt to repair myocardial tissue after

infarct, injured nervous tissues, or even in a field called organ engineering, with

the intention of generating tissues (and eventually organs) that can be implanted in

humans, eliminating tissue rejection, once stem cells could be originated from the

54

patient himself. Within that framework, mesenchymal stem cells apparently have a

wider potential of differentiation (and transdifferentiation) than the

hematopoietics, for they may be isolated from several tissues, besides the bone

marrow.

Despite the plethora of paperwork in this area, the potential of application

of those cells will only be reached after deeper knowledge of their functional and

molecular biology is achieved.

Basic Research:

As previously mentioned, the MSC stand out for their great capacity of in

vitro differentiation, being comparable to the ESCs (reviewed in da Silva Meirelles

et al., 2008). Interestingly, it was demonstrated that one type of cell related to

MSC, which is called multipotency progenitor adult cell (MPAC), is capable of

contributing to several rodent tissue when injected in the blastocyst (Jiang et al.,

2002). The great criticism to the MAPCs experiments is that these cells could be

mere artifacts of the cellular cultivation process used for their isolation.

Currently, it is known that cells having the characteristics of MSC are

distributed all over the post-natal organism. This wide distribution was assigned

due to their association with blood vessels in a model that proposes pericytes are

stem cells present in vessels (da Silva Meirelles et al., 2006). Pericytes are cells

which embrace endothelial cells in the blood vessels. Specialized forms occur in

the liver and in glomeruli. Pericytes are defined based on their relation to

endothelial cells, especially capillaries. There is evidence, however, indicating that

they form a sub-endothelial net all over the vasculature, in big and small caliber

vessels. Pericytes present a close relation with endothelial cells during the

formation of blood vessels and play an important role in the maintenance of their

structure. The pericytes markers were reviewed in a recent publication (da Silva

Meirelles et al., 2008)

Pericytes present overlapping features with MSCs such as their potential of

differentiation in vitro and in vivo, and also regarding the expression of markers.

Besides, there are studies that stress the performance of pericytes as MSCs

cartilage, bone, periodontal ligament, endometrium and fat tissue. Data like that

55

led to conception of a model where pericytes are stem cells present all over the

vasculature and act like MSCs in mesenchymal tissues (da Silva Meirelles et al.,

2006). The performance of pericytes/MSCs as active components in the tissue

repair/ regeneration process, and also in the maintenance of immune self-

tolerance was also postulated (da Silva Meirelles et al., 2008). According to that,

the tissue lesion would lead to the migration of pericytes from their niche, which

would change to an activated state and would proliferate and through the

production of extracellular matrix and molecules regulating trophic effects and

immune response, thus leading to repair of the site of the lesion. One consequence

of that is the pre-assumption that cultivated cells defined as MSCs correspond to

activated pericytes and are not, thus, equivalent to MSCs under physiological

conditions in vivo. The low homing efficiency of MSCs cultured for some passages as

compared to MSCs in primary culture (Rombouts and Ploemacher, 2003), argues

favorably to this idea.

With that in sight, the group coordinated by Dr. Dimas Tadeu Covas through

subproject 10, entitled “Basic knowledge of MSC biology by means of

comparison with pericytes”, will have as primary goal, to generate basic

knowledge about MSC biology through the comparative analysis between pericytes

and MSC. The following experimental approaches will be used: Isolation and

cultivation of human MSCs from fat tissue, umbilical cord and/or placenta, as well

as from mice tissue.

Using magnetic columns or cellular separation by fluorescence 3G5 antibody;

adipogenic, chondrogenic and osteogenic differentiation in vitro; the in vivo

differentiation of MSCs may be tested by introducing them in porous ceramic

cubes, and subcutaneously implanting them into immunocompromised mice; the

injection of freshly isolated pericytes, cultured pericytes and MSCs from Rosa26

mice into blastocysts will be performed in collaboration with Dr. Irina Kerkis at

Instituto Butantan (São Paulo, SP); the contribution of the injected cells to the

embryo will be assessed by incubation of cryosections with the substrate of the -

galactosidase enzyme (X-gal), which will allow for the visualization of the injected

cells in blue; comparison of the surface molecules expressed in pericytes, cultured

pericytes and MSC (molecules typically associated or not with MSCs, such as CD14,

CD31, CD44, CD45, CD90, CD29, CD105 and CD117, will be evaluated by flow

cytometry); cytokine profile study with immunomodulatory potential (hepatocyte


56

growth factor, transforming growth factor-β, interleukins 6, 10, 11, 15 and 27 in

fresh or cultured pericytes and MSC by flow cytometry, pericytes (fresh or cultures)

and MSC application obtained from Rose26 mice in animal model of induced

muscular lesion by cardiotoxin (the presence of muscle fibers containing the

infused cells will be observed after incubation of transversal cryosections with the

substrate for the X-gal enzyme).

The greater understanding of similarities between mesenchymal stem cells

and pericytes (Covas et al., 2008) and their close contact to endothelial cells open

important avenues about the interactions among these cell types during blood

vessel formation in response to angiogenic factor stimulation. Vasculogenesis is the

de novo formation of vessels from progenitor endothelial cells (angioblasts),

whereas angiogenesis refers to the sprounting of neovessels form a preexisting

vasculature. Angiogenic sprouting in the adult was initially considered an exclusive

feature of terminally differentiated endothelial cells, which have the capacity to

form vascular tubes and recruit pericytes.

Previous studies conducted by our group gave us the hypothesis that

immature mesenchymal stem cells reside in several peripheral tissues where they

participate in the angiogenic response to injury (Covas et al., 2008). Moreover, we

conducted studies of generation mature endothelial cells from endothelial

progenitor cells (EPC) derived from bone marrow (BM) and cord blood (CB) after

selection of specific CD133 marker expression. These CD133 positive cells from BM

and CB were induced to differentiate into endothelial cells using three different

protocols. Altogether, these studies demonstrated that vasculogenesis can be

reproduced ex-vivo by culturing BM and UCV progenitor endothelial cells with

specific cytokines and in matrigel system. Also, the role of several transcriptional

factors involved in this process could be characterized (Covas, personal

communication).

In order to continue these studies we propose (subproject 11) to investigate

the molecular interactions between bone marrow derived-mesenchymal stem cells

(BM-MSC), umbilical cord vein derived-MSC (UCV-MSC) and central nervous system

microvascular pericytes (CNS-P) after co-culture with human umbilical vein

endothelial cell (HUVEC) and ex-vivo derived endothelial cells from BM and CB. The

study of these interactions will provide relevant information concerning

physiological or pathological angiogenic and vasculogenic responses as well as


57

provide important information about vascularization process which remains one of

the major challenges in cell therapy protocols.

With this in mind, the group coordinated by Dr Dimas Tadeu Covas will carry

out the subproject 11, entitled “Investigation of angiogenic process and

molecular interactions between mesenchymal stem cells and pericytes with

endothelial cells using developed in vitro and in vivo systems” in order

determine the molecular interactions of cross-talk between FACS-sorted BM-MSC,

UCV-MSC and CNS-P with CD31 positive endothelial cells. To stimulate angiogenesis

in vitro and in vivo we will develop three models using BM-MSC, UCV-MSC or CNS-P

with endothelial cells: 1) in vitro three dimensional co-culture model of BM-MSC,

UCV-MSC and CNS-P with endothelial cells (HUVEC and BM and UCV-derived

endothelial cells); 2) the matrigel plug assay co-infused with MSC or pericytes plus

endothelial cells followed by subcutaneous injection in NODSCID mice and 3) the

treatment of acute ischemic injury in a mouse hind limb model with endothelial

progenitor or mature endothelial cells, as well as, BM-MSC, UCV-MSC and CNS-P.

For in vitro analyses of three dimensional co-culture model all cultures will

be grown for 5 to 15 days and then cells will be harvested for: a) characterization

of morphology by conventional microscopy; b) characterization of

immunophenotypic profile by flow cytometry; c) capacity to take up DiI-labeled

acetylated low-density lipoprotein (ac-LDL); d) gene expression analysis by real

time PCR; e) assessment of in vitro angiogenesis by the formation of vascular-like

structures on a matrigel system and f) characterization of new blood vessels

formed by confocal microscopy as well as by conventional microscopy after staining

the tissue formed with HE in order to determine the presence of eritrocytes

indicating functional capillaries.

For in vivo analysis two angiogenesis models will be studied: 1) the Matrigel

plug assay containing mesenchymal stem cells or pericytes plus endothelial cells

followed by subcutaneous injection in NODSCID mice and 2) NODSCID mouse

hindlimb ischemic injury produced by removal of the femoral artery. For these in

vivo models MSC or pericytes will be genetically modified with fluorescent and

bioluminescent proteins using transposon system which will allow the

characterization of new capillaries by confocal microscopy and the analysis of new

blood vessel formation in live animals using Xenogen IVIS system.


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Moreover, the interactions between these two cell types during the

angiogenesis process will be investigated using MSC and endothelial cells from large

animal models such as canine model in collaboration with Professor Maria Angelica

Miglino from Faculdade de Medicina Veterinária e Zootecnia da Universidade de São

Paulo. MSC from yolk sac canine will be isolated and characterizated because it is

the first site of blood-cell production during embryogenesis.

Recent studies conducted by Maria Angelica group have demonstrated that

the canine yolk sac contains three preferential sites for binding MSC and

endothelial cells (Miglino et al 2008). Therefore, after isolation, culture and

characterization of MSC and endothelial cells from canine yolk sac, will be

developed to a three dimensional co-culture model assess the cross-talk between

these two cell types. It should provide molecular insights into vasculogenesis and

angiogenesis process during development and tissue repair. Similarly, the same

analysis will be carried out after 15 days of culture.

The MSCs present in bone marrow and umbilical cord vein are very similar

from the phenotypic and genotypic point of view. However, a previous study

performed at Centro de Terapia Celular (CEPID - FAPESP), comparing the mRNA

expression profile by SAGE, showed small differences , indicating that the MSCs

derived from bone marrow would be more compromised to osteoblastic e

adipocytic lineages, while the ones derived from the umbilical cord vein would be

compromised to functions related to angiogenesis or vasculogenesis. If confirmed at

protein level, these differences of expression can contribute to a better

comprehension of differences between MSC of different origin and its implications

for the adequate choice for different applications in cell therapy.

Based on that exposed above, the group coordinated by Dr. Lewis Joel

Greene through subproject 12, entitled “Comparison of proteins expression of

human mesenchymal stem cells (MSC) obtained from bone marrow and umbilical

vein cord”, will have as main objective to compare the protein profile of MSCs

isolated from the vein of the umbilical Cord and bone marrow using proteomic

approach with 2D DIGE, followed by mass spectrometry, for the identification of

pre-selected proteins.

Humans mesenchymal stem cells (MSC) will be obtained from the bone

marrow, as described by Silva et al. (2003) and from the human umbilical cord vein


59

as described by Covas et.al. (2003) and Panepucci et al. (2004). The protein will be

extracted, quantified and utilized for 2D eletrophoresis. The comparison of the

“spots” profile between the different samples will be performed through the image

acquisition system Image Scanner and the program ImageMaster 4.0 (Amersham

Biosciences). The “spots” from each sample, will be cut out from the gels and the

proteins will be identified by MALDI-TOF mass spectrometry after digestion with

trypsin as described by Pereira et al. (2005). The identification will be done

automatically in the data banks of NCBI. Additionally, the list peptides mass for

each sample will be manually submitted to MS-Fit

(http://prospector.ucsf.edu/csfhtml4.0/msfit.htm) for confirmation of the

identifications done automatically. The functional classification of the identified

proteins will be performed in the Gene Ontology database (Ashburner et al., 2000)

through the FatiGO tool (http:fatigo.bioinfo.cnio.es, Al-Shahrour et al., 2004) in an

attempt to standardize the terms used to describe classes and functions of

proteins. An additional step will be the use of 2D DIGE system (two-dimensional

difference gel electrophoresis). The derivatization of proteins with fluorescent

markers will be done according to the manufacturer instructions (GE Healthcare)

and the acquisition of the gels image will be done without the removal of the gels

scanned with 3 fluorescence channels (Typhoon Trio). This step will be performed

at the Laboratório de Imunologia do Instituto do Coração (INCOR) da Faculdade de

Medicina da USP - São Paulo. The analysis of the gels will be made with the

utilization of the software DeCyder 6.0.version

In addition to hematopoietic and mesenchymal stem cells, other cells have

been shown to be potentially useful for therapeutic purposes. Amniotic epithelial

cells (AEC) can undergo in vitro adipogenic, chondrogenic, osteogenic, skeletal,

myogenic, cardiomyogenic, neurogenic, pancreatic and hepatic differentiation.

These cells express embryonic stem cell markers (SSEA-4, TRA 1-60, TRA 1-80, Oct-

4, FGF-4, SOX-2, Rex-1), unlike amniotic mesenchymal cells, which do not express

those markers. Furthermore, amniotic epithelial cells are strong candidates for

allogenic transplantations because they possess low immunogenic and

immunomodulatory properties. The great differentiation potential and the easy

access and isolation suggest that AEC can be useful and non-controversial source of

stem cells for transplantation and regenerative medicine. AEC are often cultured in

60

serum, but their clinical use requires their isolation and culture without animal

components.

Thus, the group coordinated by Dr. Wilson Araújo da Silva-Jr. in subproject

13, entitled “Differences in gene expression and immunophenotypic profile of

amniotic epithelial cells obtained with and without animal substances” will

compare amniotic epithelial cells obtained with fetal calf serum and a

subpopulation of amniotic epithelial cells, called amniotic multipotent progenitor

cells (AMC), isolated and cultured in animal substances-free conditions. Term

placentas will be obtained after delivery and amniotic membrane will be peeled

from the chorion and digested with trypsin to detach them from the adjacent

mesenchyma. Epithelial cells obtained are isolated with two different protocols:

the so called AEC are separated with a Percoll density gradient and cultivated with

fetal calf serum and epidermal growth factor (EGF). The subpopulation named

amniotic multipotent progenitors (AMP) is obtained in serum and EGF-free

conditions. Both cultivated cell are used for RNA extraction (TRIzol® LS –

Invitrogen) and to perform Microarray analysis of gene expression. Flow cytometry

will be performed to determine the expression of embryonic/epithelial markers

(SSEA-1, SSEA-3 e SSEA-4, TRA1-60, TRA1-81, NANOG e OCT-4, CD9, CD10, CD29,

CD49f, CD90, CD104, CD34, CD45, CD117 and CD140b) and immunologic surface

markers (HLA-ABC, HLA-DR, HLA-G, CD80, CD86, CD40, CD40L, PD-1, PD-L1, PDL-2

e HLA-G). The ability to differentiate into all three germ layers will also be

compared. According to Miki and colleagues (2005), RNA will be extracted from

both types of cells and gene expression profiles will be evaluated by microarray

analysis (Agilent Technologies).

Although, stem cells are isolated on the basis of the expression of cell-

surface markers together with the well known plastic adherence property, more

primitive stem cells can be identified on the basis of the their ability to effluxe

Hoechst 33342 dye. As a consequence, these cells acquire the profile of low side

scatter and bright Hoechst staining cells, which is termed “side population”

(Goodell et al.,1996). Originally, side population phenotype was described in

murine bone marrow preparations which are present at frequency of 0,05%.

Moreover, the SP cells have a greater ability to reconstitute hematopoietic system

compared to HSC-CD34+ obtained from bone marrow. The presence of SP cells was



61

also demonstrated at human bone marrow, as well as, other tissues such as, liver,

brain, mammary gland, kidney, skin, testis and retina (reviewed in Challen e Little,

2006). These data suggest that SP might be widely distributed into several tissues;

although functional role and characterization are not well understood.

Previous studies conducted by our group demonstrated the presence of SP

cells in human bone marrow which express high level of pluripotent gene markers,

such as, Nanog, Oct4, c-myc e b-catenina (Picanço-Castro, personal comm.).

Moreover, since embryonic stem cells present two important application

limitations: a) immunological rejection and b) tumorigenic potential, the use of SP

cells can constitute an alternative safe cell source for autologous clinical

applications. The group of researchers from the Center for Cell-based Therapy has

been developing research projects with mesenchymal stem cells for the last eight

years. Indeed, we were the first to demonstrated the presence of MSC in umbilical

cord vein (Covas et al., 2003) and recently we showed that MSC are widely

distributed into the organism and present extensive similarities with pericytes

(Covas et al., 2008).

In view of the importance of identifying and characterizing more primitive

stem cells followed by the evaluation of their therapeutic potential, the group

coordinated by Dr. Dimas Tadeu Covas will carry out the subproject 14, entitled

“Isolation and functional characterization of pluripotent stem cell - side

population”, whose aim is to isolate the side population from two human tissues

(umbilical cord vein and central nervous system blood capillaries) and to compare

then with SP isolated from bone marrow. The second aim of the subproject 14 is

the isolation and characterization of human SP obtained from mesenchymal stem

cells expanded in culture and isolated from bone marrow, umbilical cord vein, as

well as, brain pericytes expanded in culture. In parallel, SP cells isolated from

human bone marrow, umbilical cord vein and brain capillaries at day 0 will be

submitted to expansion in culture using four different medium conditions: a)

culture medium for MSC expansion (MSC-M); b) culture medium for expansion of

hematopoietic stem cell (HSC-M); c) culture medium for expansion of endothelial

progenitor cells (EPC-M) and d) culture medium for expansion of human embryonic

stem cells. After expansion SP will be characterized in terms of morphology,

immunophenotying and multipotentiality features. Also, the gene expression profile


62

will be determined by RT-PCR for the functional characterization of these different

sources of SP cells. Finally, the third aim of the subproject 14 is to investigate the

therapeutic potential of cell-derived SP cells using murine model for hepatic

endothelial injury, as well as, for hind limb ischemia injury.

In collaboration with the group of Professor Dra Maria Angélica Miglino the

fourth aim objective of the subproject 14 consists of isolating SP from ovine bone

marrow and brain capillaries, as well as, from canine yolk sac. The number of SP

cells will be analyzed by flow cytometry and clonogenic assay. Next, these SP will

be grown and expanded in three different mediums a) MSC-M; b) HSC-M and c) EPC-

M and the therapeutic potential of some of these SP cells will be investigated in a

model of canine bone marrow aplasia and ovine brain ischemia injury.

A second collaboration with the group of Professor Dr. Flávio Vieira Meirelles

to isolate SP from two bovine/GFP+ fetal tissues consists in the fifth objective of

this subproject. The bovine fetal GFP tissues, the bone marrow (BM) and umbilical

cord vein (UCV) will be collected in utero. After the isolation, the SP cells will be

grown and expanded under three specific medium cultures: a) MSC-M; b) HSC-M

and c) EPC-M and the therapeutic potential of some of these SP cells will be

investigated in the NODSCID murine bone marrow transplantation model. In

addition nuclear transfer experiments will be performed in bovine enucleated

oocytes using BM or UCV-SP/GFP+ cells in order to evaluate the genetic

reprogrammation ability of SP cells. Also, in collaboration with Prof Dr. Flávio the

nucleus of human SP cells will be used for genetic reprogramming studies using a

bovine cytoplast as receptor.

The use of the entire potential of stem cells in therapies directed to repair

of some tissue, depends on the deep understanding of the mechanisms that control

the processes. Mesenchymal stem cells .(MSCs) are capable of differentiating into

multiple cell types, however,the knowledge of the paths and molecules that

regulate the maintenance of the undifferentiated state, as well as the

differentiation process, are not clear. In this last decade, special attention has

been given to the noncoding RNAs, since they are considered to be important

elements related to the endogenous regulation system. The miRNAs compose a

small group of these endogenous RNAs, containing 18 to 25 nucleotides. In humans,

500 miRNAs have already been identified (GRIFFITHS-JONES et al., 2006). The



63

miRNAs seem to have several biological functions, such as in the insuline secretion,

hematopoiese, embryonic muscle development and maintenance and

differentiation of stem cells (BARTEL, 2004; DU & ZAMORE, 2005; WIENHOLDS &

PLASTERK, 2005). They also participate in the functional aspects of cells, such as

proliferation, survival and apoptose (DI LEVA et al., 2006). The expression of the

miRNA in multicellular organisms is not just temporal, but also specific in some

tissues, implicating that they have an essencial role (BARTEL, 2004; DU & ZAMORE,

2005; WIENHOLDS & PLASTERK, 2005). Some author investigations have already

demonstrated that microRNAs (miRNAs) are unregulated in situations like cancer, in

viral and genetic diseases, in the maintenance and differentiation of adult and

embryonic stem cells, among others. Recently, Mizuno e cols. (2008) demonstrated

the reduction of the miR-125b expression during the osteoblastic differentiation

process in murine MSCs. Up to the present moment, no miRNA has been reported to

be an important factor in the differentiation of human MSCs .

Thus, the group coordinated by Dr. Dimas Tadeu Covas in subproject 15,

entitled “Functional evaluation of microRNAs in mesenchymal stem cells

differentiation”, will have as general objective, the evaluation of the profile of

the expression, as well as the role of miRNAs during the differentiation of MSCs

into osteocyte. The following experimental approaches will be taken: to isolate and

cultivate bone marrow MSC; to promote the differentiation into osteocyte and

collect samples in different times: day zero, 24 hours, 72 hours, 7 days, 14 days

and 21 days; to quantify the level of expression of mature miRNAs by using the

TaqMan® microRNA assays Human method and select those potentially involved;

analise the functional effect of selected miRNAs by reducing their levels making

use of the Anti-miR™ miRNA Inhibitor (Ambion) system; to analise the functional

effect of selected miRNAs selecionados by redicing their levels through the Pre-

miR™ miRNA Precursor Molecule (Ambion) system; to detect potential target genes

for miRNA-125b using algorithms such as miRanda and RNAhybrid.

The capacity of self-renewal, as well as the potential of differentiation in

different cellular types of different tissues, make the adult stem cells a promising

tool for the study of certain diseases. Moreover, the ease of cultivation of the CTM

and their capacity of differentiation into osteoblasts allow that those cells be used

as model of study for bone disease. The osteogenesis imperfecta (OI), one of the

most frequent dysplasia, occurs equally among all ethnic groups and is charcterized


64

as a genetic disorder of mesenchymal cells, in which a generalized osteopenia leads

to bone deformity, excessive bone fragility and short stature. The main

characteristics of the disease are from the insufficient or defective production of

colagen molecules. Some studies have been conducted with the objective of

identifying genes with differentiated expression during the osteogenic

differentiation process. However, there are few functional studies of differentially

expressed genes in cells from patients presenting OI. This way, the group

coordinated by Dr. Wilson Araújo da Silva Jr, through subproject 16, entitled

“Gene expression profile of mesenchymal stem cells of patients with

Osteogenesis Imperfecta during in vitro osteogenic differentiation”, will analize

the profile of genic expression in mesenchymal stem cells during the differentiation

process in osteoblasts of bearers of Osteogenesis Imperfecta and of control

individuals. For that objective, we propose: the cultivation and expansion of

mesenchymal stem cells from patients and normal donors; analysis of genic

expression in large scale making use of microarrays; analiysis of bioinformatic for

the identification of gene the networks, which are fundamental for the

osteogenese; analysis of the expression by Real-Time PCR; functional studies of

gene interference using RNAi.

The systematic localization of CTMs in the walls of all blood vessels

associated with their imunnossuppress e imunoregulator properties raise questions

about the eventual participation of these cells in inflamatory auto-imune diseases

(AID).There are few studies about the genetic and functional characteristics of the

CTMs of patients presenting AID’s and no data about the animal model CTMs and

the genetically determined AID’s . Furthermore, it has not been established if the

CTMs of patients having AIDs or genetic animal models of AIDs are normal or

defective cells. The diabetes mellitus type 1 (DM-1) and the multiple sclerosis (MS)

are mediated by self-reactive T cells, and characterized by the pancreatic β cells,

which produce insulin and the central nervous system, respectively. In addition the

susceptibility to DM-1 and to MS is determined by genetic and environmental

factors. Thus, the molecular characterization of the CTM obtained from these

patients may reveal both altered genes and sinalization path ways and/orfunctional

efects, possibly related to the initiation of autoimmune and or the pathogenesis of

the diseases which, in the future, can be throroughly studied will facilitate the

development of new therapeutic reassures for the treatment of AIDs. On the other


65

hand, if the CTMs from patients with MS to be genetically and functionally normal,

it will be possible to use them as source autologous cells for the treatment of

patients with DM-1 and MS.

The group coordinated by Dr. Júlio César Voltarelli, through subproject 17,

entitled “Genic, proteic and functional analysis of mesenchymal stem cell (MSC)

in patients with auto-immune disease”, will evaluate the functional characteristic

and the profile of gene and protein expression on large scale of CveTMs of patients

with DM-1 e EM. For that, 15 patients with DM-1 and 15 patients with MS will be

studied and submitted to the treatment with HDI/AHSCT, at the Unidade de

Transplante de Medula Óssea do Hospital das Clínicas da FMRP-USP. Bone marrow

samples of the patients with DM-1 or EM before the HDI/AHSCT and six months

after the transplantation. Samples of BM of healthy individulas will be collected

(bone marrow volunteer donors) for isolation of MSCs or control of the

experiments. The mononuclear cells of the BM of the patients will be isolated by

density gravity using centrifugation for isolation and culture of MSCs, and at the

third step, samples of MSCs will be separated for: 1, biological characterization of

MSCs (CFU-F, immunophenotyping, proliferative capacity); 2, evaluation of the

immunosuppressant capacity of the MSCs in vitro (lymphocyte proliferation

inhbitions assays by the co-culture with MSCs); 3, adipocytes and osteocytes

differentiation; 4, RNA extration to the gene expression analysis by large scale

microarray and RT-PCR validation; 5, protein extration to the protein expression

analysis by 2D eletrophoresis.

Subproject 18: “Determination of some proteins related to apoptotic process and

cell cycle control of hematopoietic and leukemic progenitors”

The new chemtherapy schemes developed for acute myeloid leukemia (AML)

have induced the remission of the disease and increased the survival of leukemic

patients. However, relapses are a very common especially among older patients

and/or patients having a bad prognosis. The relapses were attributed to the

existence of quiescent leukemic stem cells resistent to chemiotherapy. The

leukemic cells, the origin of normal precursor cells are similar to those though, not

identical, because genetic events such as mutations and chromossomic

translocations were responsible for their neoplasic properties . Several studies have

demonstrated that differences in the level of RNA and proteins involved in



66

apoptosis and control of the cellular cycle are demonstrable when leukemic and

normal cells are compared. Differences in the protein levels were also

demonstrated when leukemic cells were treated with the trans-retinoic (ATRA) and

As2O3. The purpose of this research is to identify the differences in the level of

proteins of apoptótico interest in normal hematopoietic stem cells

(CTHs)(CD34+CD38-) and in leukemic stem cells (CTLs) (CD34+CD38-CD123+). The

comparison between the expression level of non or treated blasts with As2O3 will

also be made. The proteins to be studied are: Electron transfer flavoprotein beta-

subunit, antioxidant 2 protein, HMGB-1, c-H-ras, Anexin I, Peroxirredoxin-2, Rho-

GDI alfa, Rho-GDI beta and also three proteins described in the apoptose pathways:

caspase-3, caspase-9 and BCL2. The nucleofosmina-1 protein, of major interest in

the control of the cellular cycle will also be monitored at the present study. A

selective proteomic approach for proteins related to the apoptosis will be used in

this work, which does not represent an inventory of proteins of leukemic cells. The

proteins involved in the apoptotic processes and control of the cellular cycle

described in the literature, whenever possible, will be identified by

immuneological methods and confirmed by when possible by mass spectrometry of

tryptic peptides. The proteins will be separated by 2D electrophoresis. We hope to

obtain a profile of apoptotic proteins in leukemic stem cells and also in the

leukemic blastocytes treated with As2O3.

Dr. Eduardo Magalhães Rego’s group in the subproject 19 entitled

“Evaluation of the function of dyskerin in early hematopoietic differentiation”

will study the mechanisms involved in the malignant transformation of the

hematopoetic stem cells using two animal models: a) mutant mice for the DKC1

gene and b) an acute leukemia model using vervet monkeys transplanted with

hematopoetic stem cells retrovirally transduced with the CALM-AF10 fusion gene

(subproject 20). The DKC1 gene encodes dyskerin (Heiss et al., 1998), and is

characterized by multiple pathological features, including increased susceptibility

to cancer and bone marrow failure (Dokal. 2000). Pancytopenia associated with

hypocellularity of the bone marrow is the hallmark of severe aplastic anemia, a life

threatening disease. The associated DKC1 mutations X-DC is one of the major

inherited aplastic anemia syndromes. However the cellular and molecular

mechanisms leading to bone marrow failure in dyskeratosis congenita are unknown.

The DKC1 gene encodes for a pseudouridine synthase that modifies ribosomal RNA



67

(rRNA) and is essential to ribosome biogenesis. Therefore, if the mechanism leading

to aplastic anemia and cancer in X-DC is linked to the impairment of ribosomal

function, it would be the first demonstration of the role of this organelle in the

fate of immature undifferentiated progenitors.

The specific objectives are:

1) To examine the role of dyskerin in hematopoietic stem cells. In order

to determine whether the abnormalities in Dkc1m mice is due to a

autonomous cell defect, we will perform competitive repopulating

assays. In these experiments, hematopoietic stem cells from Dkc1

mutants will be tested against their wild-type counterparts in their

capacity for repopulating lethally irradiated recipients. If Dkc1 function

was essential to the hematopoietic stem cell, one should expected the

reduction in number of mature cells from all the tested cell lines

(lymphoid, granulocytic/monocytic and megakaryocytic) of Dkcm mice.

2) To examine the role of dyskerin in early lymphoid differentiation.

Based on our preliminary results, the best candidate to be the

hematopoietic progenitors targeted by Dkc mutations are the Common

Lymphoid Progenitors (CLPs). We will isolate CLPs from Dkc1m and wild

type controls, and perform competitive repopulating assays as described

above, but as recipients we will use rag-/- mice, which do not present

mature T or B cells in the peripheral blood.

3) To quantify the expression of the Dkc1 gene in isolated progenitors.

We plan to isolate HSCs, CLPs, Fractions A and B of lymphopoieses,

common myeloid progenitors (CMPs), Granulocyte-Monocyte Progenitors

(GMPs), and Myeloid-Erythroid Progenitors (MEPs) from the bone marrow

of Dkc1m and wild-type mice by Fluorescence-activated cell sorter

(FACS), and to measure the expression of the Dkc gene by Real-time PCR.

4) To analyze apoptosis and cell growth of Dkcm lymphoid progenitors.

Once the specific hematopoietic progenitor in which Dkc is qualitatively

and quantitatively defective is identified. We will test whether it is more

susceptible to apoptosis and/or may harbor defects in cell growth

regulation compared to their wild-type counterparts.

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Subproject 20: “Acute Leukemia Model in Chlorocebus aethiops

(Cercopithecus aethiops) using haematopoetic stem cells transduced with

retroviral vector containing the hibrid gene CALM-AF10”

In acute leukemia research, the animal model provides essential subsides for

the understanding of molecular and physiological mechanisms involved in leukemia

development. In subproject 20, we propose to generate a model of human acute

leukemia using the vervet monkey. Despite the fact that the mouse model is the

most commonly used, because of its relatively low costs and easy housing and

manipulation, it has some limitations. From the experimental point of view, some

techniques are not feasible due to the size of the mouse, making the serial

sampling difficult. Genetically, the murine model differs from the human in many

aspects, leading to the need of result validation in a phylogenetical closely related

organism for pre-clinical studies. The vervet monkey (Chlorocebus aethiops) is one

of the most used non-human primates in biomedical research (1). The Chlorocebus

aethiops became an important model for the HIV study, once it is easily infected by

the simian immunodefficiency virus (SIV) but differently than the Rhesus monkey,

the Chlorocebus aethiops does not develop the disease (2, 3). The

t(10;11)(p13;q14), involving the CALM e AF10 genes has been reported in acute

lymphoid, acute myeloid leukemia and malignant lymphoma (7, 8, 9). It is involved

with a bad prognosis disease, similar to the leukemias with involvement of the MLL

gene (10). Leukemic cells from CALM-AF10 patients and mice co-express myeloid

and lymphoid antigens, making these cells an important for the study of leukemia

stem cells (11).

The need for an animal model close to the human for translational and pre-

clinical studies for leukemia and stem cell research, the availability of Chlorocebus

aethiops at the National Primates Center (CENP) and the presence of a group of

investigators with experience in the proposed models makes the proposed project

feasible. The aims of the subproject 20 are:

1) Standardization of blood and bone marrow sampling at the CENP

laboratory. Evaluation of hemogram, myelogram and cytogenetical

parameters of Chlorocebus aethiops, establish reference values for the

species in the maintainance facility. The extent of genetic homology



69

between human and Chlorocebus aethiops will be researched in parallel

for relevant genes using bioinformatic tolls.

2) Immunosuppressive regimen determination with myelosuppressive

drugs or irradiation at the CENP laboratory. The bone marrow stem

cells transduction protocol will be established using the conditioned

medium with viral supernatant (VCM) containing the retroviral construct

with the CALM-AF10 fusion gene and the reporter gene GFP or YFP, and

the empty vector containing only the reporter gene. The transduced cells

will be analyzed in vitro for clonogeneicity and transformation. In case of

transformation, the effect of CAPE on the cell growth will be analyzed.

The transduced bone marrow cells will be transplanted back to the donor

after myeloablative treatment.

3) Cytogenetic and gene expression profile of the leukemic cells. The

receptors will be monitorized for any clinical/hematological/cytogenetic

alteration. The gene expression profile of these cells will be analyzed.

4) Treatment with experimental drugs. Leukemic animals, as well as

leukemic cells from the animals, will be treated with experimental

treated with experimental drugs, such as cafeic ester (CAPE).

The group coordinated by Dr. Júlio César Voltarelli will carry out subproject

21,entitled “Evaluation of the therapeutic potential of mesenchymal stem cell

(MSCs) from skin lesions caused by extensive thermal burns in animal model”,

will study the therapeutic potential of xenogenic and alogenic MSCs in the

treatment of ulcers caused by extensive thermal burns in rats. Since Gal-1 takes

part in the process of tissue regeneration and is highly espressed in CTMs, we also

intend to study the impact of Gal-1 over the therapeutic potential xenogenic MSCs

in the treatment of ulcers caused by burnt.

MSCs will be isolated from the bone marrow of mice C57BL/6 wild (Gal-1+/+) or

without the gene Gal-1 (Gal-1-/-) or from the rats and in vitro expanded. The MSCs

(alogenics, xenogenic, wild or Gal-1+/+) and/or murine recombinant galectin-

1(Gal-1rm) wil be topci or systemic administratedc, animals previously submitted

to extensive burning of their skin. After the treatment of the burns with theCTMs,

or not, the following will be evaluated: the gravity of the burn, the process of



70

tissue regeneration, the state of systemic immunosuppression, the expression of

angiogenic factors, growth factors, chemocinas and cytocinas in byopies of the

ulcer and differential protein expression by proteomic methods. The action of

mechanisms CTMs in the regeneration process of ulcers caused by extensive burning

will be studied in this project. In addition to that, a comparison between the

therapeutic potential of CTMs topically or systematically via and a comparison

between the therapeutic potential of CTMs.

The ability of mesenchymal stem cells (MSC) to differentiate into several cell

types in vitro, their relative ease of expansion in culture, their immunologic

characteristics, and their ability to be genetically modified maintaining the

multipotent features, clearly make MSCs a promising source of stem cells for tissue

repair and gene therapy. In this sense, the group coordinated by Dr. Dimas Tadeu

Covas will carry out subproject 22,entitled “Use of mesenchymal stem cells

genetically modified with human recombinant coagulation factor IX in

Hemophilic B mice” with the aim to determine the suitability of developing a

hepatic MSC cell line to produce the recombinant human factor IX for the

treatment of hemophilia B in a mouse model. Hemophilia B is a genetic disease

caused by the mutation in the gene encoding blood coagulation IX, which is located

on the X chromosome. Hemophilia B occurs in about 1 in every 20,000 males, and

the current therapy consists on intravenous administration of the clotting factor

concentrates obtained from blood donors.

The two major limitations to present day hemophilia therapy are: 1) viral

contamination risks and 2) the cost, which limit their use to a small group of the

global haemophilia population (Lillicrap et al., 2006). On the other hand, two

facts: 1) the pathology involves a single protein and the replacement of this protein

through cellular or genetic substitution should and does indeed eliminate the

disease manifestation and 2) relatively small increments of clotting factor levels (1-

5% of normal) have been shown to result in significant clinical benefits in the long-

term have stimulated researchers to develop several gene therapy strategies for

treatment of Hemophilia B. Among pre-clinical studies it can be cited: 1) gene

therapy protocols which consist on the infusion of recombinant viral vector

producing human factor IX and 2) cell therapy protocols which consist on retroviral

transduction of autologous cells (skin fibroblasts) followed by infusion into

haemophilic models (reviewied in Lillicrap et al., 2006).


71

Recently, two clinical trials have been approved by FDA. Both include the

use recombinant adenovirus producing hFIX. The major limitations of this strategy

are the adverse host immune responses to these vectors and the high demand of

viral particles due low cell production level.

The research group of Hemocentro de Ribeirão Preto has large experience

with gene modification of stem cells and mammalian cell lines using retrovirus

system. Moreover, the group develop research projects in this field since 11 years

ago which resulted in the generation of a recombinant cell library containing 43

transgenic cells producting VIII and IX coagulation factors. Three of these cell lines

produce high level of recombinant factor IX. Also, the group develops research

projects with mesenchymal stem cells since 8 years ago and recently designed a

clinical protocol to treat patients who developed the graft versus host disease after

bone marrow transplantion. The clinical trial has been approved by the Research

Comite and the program has been started. With this in mind, the proposal of this

project consists on generating a cell therapy strategy for hemophilia B in a murine

model.

The increasing number of clinical applications has stimulated a need for

alternative to rapidly expanding MSCs. The actual methodology for mesenchymal

stem cell culture presents four main limitations: 1) excessive manipulation which

can interfere with functional cell proprieties due to successive passages employing

enzymatic treatments; 2) high contamination risk recurrent from extensive

manipulation; 3) absence of culture control parameters and consequently absence

the control of cell physiology control and 4) high cost and time-consuming

procedures for generation of sufficient number of undifferentiated cells of

adequate quality. This proposal involves the technology development for

optimization of MSC culture system in bioreactors aiming to reduce one of the

limitations cited above and to promote the development of a scale-up with tighter

control of culture conditions in order to provide a clinical grade product in an

efficient, economic and safe manner. The stem cell culture in bioreactors using

microcarriers, to be efficient in terms of the expansion and maintenance of original

phenotype (Schop et al., 2008; Frauenschuh et al., 2007; Malda et al., 2006).

Futhermore, some types of microcarriers provide an available cell growth area of

about 20 cm2/mL compared with conventional monolayer cultures are 4 cm2/mL.

72

With that in mind, the group coordinated by Dr. Dimas Tadeu Covas in

subproject 23 entitled “Bioprocess development for mesenchymal stem cell

expansion on microcarriers”, will have as its main objective, the development of

a bioprocess for culture and expansion of mesenchymal stem cells from bone

marrow and umbilical cord in microcarriers and the evaluation of the biological and

functional proprieties post-culture. The methods involved in this project include

mononuclear cells separation and culture and expansion of mesenchymal stem cells

from bone marrow blood and umbilical cord; MSC culture in spinner bioreactor on

commercial microcarriers (Cytodex 3 - GE Healthcare and Cultispher S - Percell

Biolytica) with dissolved oxygen, pH and stirring control; morphological analysis

employing phase contrast and conventional microscope after coloration by

Leishman; analysis of imunophenotypic profile by flow cytometry using 18

monoclonal antibodies (CD105, CD73, Stro-1,; CD90-PE,CD29, CD13, HLA ABC,

CD49e, CD54, CD44, CD51/61, CD106, CD34, CD14, CD45, HLA-DR, CD31, KDR). The

cytogenetic profile obtained using classic cariotyping protocol using GTG banding,

evaluation of differentiation potential in vitro (stain by SudanII/Scarlat for

adipocyte, von kossa and alkaline phosphatase for osteocyte, hematoxilin-eosin for

chrondocyte and immunostain with anti-type II collagen; and quantitation of gene

expression by Real-Time RT-PCR using SYBR green chemistry and TaqMan PCR

assay.

Thus, the group coodinated by Dr. Jose Cesar Rosa with the subproject 24,

called “Functional Proteomic: the role of nucleophosmin (NPM) in gliomagenesis”,

has as main objective study, through proteomics methodology, the NPM role in

T98G and U87MG cell lines, derived from multiform glioblastoma (grade IV. To do

that, the changes in proteome will be relacionated with proliferation, cell

migration, and apoptosis apoptose under EGF stimulation and its effect in these

protein level, as well, as the NPM expression silencing by interference RNA (iRNA).

Consequently, we will be able to infer, by the proteomic methdology, other

proteins up or down-regulated that will be directly related to the NPM expression.

We also propose an extension of our proteomic studies of glioma to additional 5

patient sample of each grade using 2D gel electrophoresis/MALDI-TOF/TOF-MS and

complementation by shotgun peptide sequencing (SPS) which should contribute to

increase the number of proteins candidates for new biomarkers or targets for

therapeutic intervention.



73

Chronic lymphocytic leukemia is characterized by progressive lymphocytosis

of small mature B cells, imunophenotype CD5+/CD19+ and accumulation of cells in

blood, bone marrow and lymphoid organs (Caligaris-Cappio e Hamblin, 1999). CLL

represents 40% of all leukemias in adults over 65 years old. Only rare cases occur in

patients below 30 years old. In addition to the mutational status of variable region

of the immunoglobulin heavy chain (IGHV), and expression of ZAP70 and CD38, DNA

copies number variation are found in 50-80% of CLL patients and are considered

important prognostic markers of the disease (Doneda, Montillo et al., 2003). The

most common chromosomal abnormalities seen in CLL are the trisomy 12 and

deletions in 13q14, 11q22-q23 and 17p13. Among them, the del (13)(q14) is

associated with a favorable prognosis and the other chromosome losses are

associated to a unfavorable prognosis (Dohner, Stilgenbauer et al., 2000).

Although several genomic regions involved in chromosomal changes have

been described in CLL, relatively few onco and tumor suppressor genes have been

involved (Tyybakinoja, Vilpo et al., 2007). The combination of cytogenomic

methods, (spectral karyotyping and arrayCGH) will allow the identification and

characterization of the break points, associated with chromosomal changes,

detection of changes in the number of DNA copies and identification of possible

involved genes. The analysis in high resolution will identify submicroscopy

deletions, which will be informative, especially in patients with normal karyotype.

Thus, the group coordinated by Dr. Roberto Passetto Falcão with the

subproject 25, named “Cytogenomic tools applied to the investigation

of chromosomal instability in chronic lymphocytic leukemia (CLL)”, this Project

has the aims: to evaluate the genomic profile of the LLC, through

cytogenetics molecular tools, as the spectral karyotyping (SKY) and arrayCGH to

better define and characterize genomic regions and possible genes involved.

Additionally, the project aim to create a bank of samples from patients with CLL

(cells, DNA and RNA) to be used for the development of other research projects.

For that reason, the following methods will be employed: obtaining cells

for SKY technique application; metaphasic cells hybridization for spectral analysis

(Spectral Applied Imaging); extraction of DNA from blood of patients and

controls and changes evaluation in the number of genomic copies regions

(duplications or deletions) by the arrayCGH method using Agilent platform.


74

Estabishment of Animal Models and Pre-clinical trials

Embryonic and adult stem cells research described in this Project cover the

methodology establishment and questions of important studies, in a conceptual and

practical order. Together, forms the development base ot technical-scientifically

know how necessary for the stem cell therapy establishment, as a future reality in

Brazilian medicine. However the effective practical application of these potential

cells demonstrated in vitro or in laboratory animal’s trials, will must depends of

new clinical trial with animals with more close characteristics of human organism.

In this way, we describe a propose of effective implantation of a Centre capable to

base studies in this nature.

In front of the establishment necessity of expertise on the applicable

biotechnology field of cell and gene therapy in animal models, the infrastructure

adaption for new technologies development and training program of students is

fundamental to guarantee the quality of the results of this project. Then, last years

ago, different initiative must be done to create a Center of Pre-clinical trials and a

Stem Cell bank of animal models, centered locate at Faculdade de Medicina

Veterinária e Zootecnia of USP centered with the establishing of a core facility

system located at USP and wide-range throughout the Satellite Laboratories Net

associated to Pre-clinical Studies Center.

With great importance, this Center open new venue to establish pré-clinical

trials in animal with high disease incidence common in human, neurodegenerative

disease, spinal cord lesions and bone defects, and also, disease of the eyes.

The group coordinate by Dra. Maria Angélica Miglino related to subproject

26, titled “Pre-clinical studies center and animal stem cells banking”, Will have the

main objective to structured a bank of animal stem cells with high level

competence related to Regenerative Veterinary Medicine. Different protocol will

be used in various pathology and the procedures collection of stem cells in na idela

medium for expansation, analyses of potential differentiation, protocols, transfer

of pre differentiated cell to a surgical area, byopsie and biodistribuition and

engraftment of stem cell inside the damage tissue

Follow the list of animal models that will be used and cell types related to

each disease, in a partner work between groups.



75

Acquired and/or induced-diseases in animal models like:

Species: canine, suine, ovine, equine, rabbits, mouse, rats, hystricomorphs and

monkey;

Diseases: bone marrow aplasia, acute kidney lesions, head femur osteonecrosis,

corneal and retina processes and, cardiac and pulmonary diseases;

Criate by animal stem cells banking (culture and differentiation);

Canine mesenchymal cells from yock sac, boné narrow na fetal liver

Equine mesenchymal cells from umbilical cord e amnion;

Progenitors cells by canina olphactory epitelium

Stem cells from amniotic flui, yolk sac e alanttois

Experimental topics

Produce positive LACZ mesencymal cells from umbilical cords and bone narrow to

autologous transplantation

Intra-uterine injection and biodistribution of the canine mesenchymal stem cells;

Biodistribution of mesenchymal stem cells and GFP+ precocities in ovine embryos;

Culture and differentiation from monkey mesenchymal stem cells - Callitrix

(collaboration to Maria de Fátima Messias and Penelope Nayund, Germany).

The large animal models definitely contribute for the cell therapy protocols

establishment, permitting that clinical procedures near to the utilized in humans

are developed. However, the real transference for humans, can depend on

behavior and biological characteristics that are human exclusivities or. at least, of

animal near from them on evolutive process. Primates not-humans are considered

good experimental models in biomedical researches because they present anatomy

and immunologycal system with very similar answers them found in humans (CCAC,

1993). Despite of this characteristics, researches related to primates, not human,

using cells are still in smaller number compared to those developed in others

animal models as rat and mice, this fact is because it is more difficult to access

76

and handle primates not humans in researches (Jin et Al., 2002; Hodge Jr., 2002;

Takagi et Al., 2005; Vrana et Al., 2002). Because of this, the availability of a

plantel of primates not humans and maintained in captivity for experimentation

biomedical researches, would be able to contribute hugely for the advancement of

the researches that utilize stem cells for treatment of diverse illnesses. In this

sense, the National Center of Primates - Evandro Chagas Institute (CENP-

IEC/SVS/MS), Ananindeua, Pará, is going to dispose the experience already

established in management, clinical and surgery of primates not humans

maintained in captivity, for the utilization in biomedical researches using stem

cells.

Like this supracited group, the group coordinated by Dra. Klena Sarges

Marruaz da Silva with the subproject 27, titled " To make Neotropical primates

(new world) and Chlorocebus aetiops (old world) to stem cell research as animal

model for cellular therapies ", will have as principal objective, to dispose the

plantel of neotropical primates neotropicais and Chlorocebus aethiops, in adult

ages, born in the National Center of Primates - Evandro Chagas Institute (CENP-

IEC/SVS/MS), to use adult and embryonic stem cells with purpose of cell therapy.

For that, adult males of Chlorocebus aethiops specie (or of other neotropical

species) will be selected by the veterinarians of the CENP-IEC/SVS/MS through

clinical selection where will be carried out clinical and laboratory exams (blood

count, serum biochemical, urinalisys, parasitologic analyses of excrements and X

ray). Only the clinically healthy animals will participate of the experimental

groups. The animals selected will be separate. Them the cellular cultive prepared

for transplant will be transported from the university or partner center to the

laboratory of cell cultive of the Section of Environment/IEC/ SVS/MS, where will be

stored, and, to the epoch of the transplants, caused to the CENP-IEC/SVS/MS, them

these cells will be achieveted until the experimental phase with the animals. The

transplant of the cultive will be done according to experimental protocol

established for each experiment in anesthetized animals, by a veterinarian of the

CENP-IEC/SVS/MS according to the protocol already established. The procedures

will be done in aseptic conditions, and the trichotomized region, cleared with

antiseptics, will receive the cells or placebo. For the clinical accompaniment after-

transplant, the animals will be evaluated through complete blood counts (CBC),

with white blood count (WBC) and also by clinical evaluations for corporal


77

temperature, weight or any another exam suggested according to the kind of

transplant used. For histopathologic evaluation of the main organs (heart, lung,

kidneys, liver, spleen, linfonodes, etc) and bone marrow, the euthanasia will be

done in one animal of each group participant of the experiment.

Clinical Studies

In spite of the small number of clinical studies aimed at the evaluation of

stem cell based therapeutic approaches, the use of adult stem cells to treat some

diseases is a reality. Thus, the group coordinated by Dr. Júlio César Voltarelli is in

the vanguard of autoimmune diseases treatment in Brazil and worldwide. In

addition, the group led by Dr. Dimas Tadeu Covas is in the forefront of the use of

autologous mesenchymal stem cells for the treatment for graft-versus-host disease

(GVHD). Due to their importance, they will be discussed in details:

Subproject 28: Treatment of type 1 diabetes mellitus by infusion of

mesenchymal stem cells

Abstract

The type 1 diabetes mellitus (DM-1) is an inflammatory autoimmune disease

(AID), mediated by autoreactive T cells and characterized by the selective

destruction of insulin-producing β cells. The susceptibility to DM-1 is determined by

genetic and environmental factors. The clinical disease expresses itself only after

the destruction of approximately 80-90% of the β cells. The destructive process of β

cells leads to the lack of insulin, which results in hyperglycemia and its acute and

chronic complications. If not treated, the metabolic consequences progressively

lead to depression of the central nervous system, coma and death. Thus, patients

need permanent treatment with exogenous insulin for survival. Among other

serious chronic complications are the vascular problems that lead to renal failure,

blindness, heart disease and chronic ulcers.

DM-1 is treated by conventional or intensive insulin therapy. Clinical tests

with immunosuppressive drugs, monoclonal antibodies (anti-CD3) and islet

transplantation have had significant results, but they have been insufficient for

routine clinical application. This scenario stimulated new studies concerning to

therapeutic alternatives, such as in vitro differentiation of human stem cells into


78

pancreatic β cells for transplantation and treatment. In this context, mesenchymal

stem cells (MSCs) represent an ideal source for cell therapy due to their ease of

isolation and in vitro expansion, and also by their immunsuppressor,

immunoregulators and regenerative capacities.

Recent studies have demonstrated a suitable therapeutic effect of MSCs

infusion into animal models of autoimmune diabetes and in patients with

manifestation of GVHD, whose immunologic manifestations are similar to those in

autoimmune diseases. On the basis of published studies, our hypothesis is that the

autoimmune aggression over the pancreas can be controlled through the MSCs

because after infusion in diabetic patients, they preferentially migrate to the

inflamed pancreatic tissue, promote local immunosupression and stimulate the

local regeneration through paracrine mechanisms that involve the production of

anti-inflammatory molecules, imunorregulators, growth factors and angiogenic

molecules.

Thus, our objective is to evaluate the safety, the therapeutic effect and the

mode of action of the infusion of allogenic MSCs in the treatment of recently

diagnosed DM-1 in humans and animal models. We will evaluate the immune

response (frequency of T cell subpopulations, naïve T cells, memory T cells, NK

cells and CD4 and CD8 T cells; cytokines measure at serum, T cell repertoire,

autoreactive T cells, and anti- β cells) in patients at different time after the

infusion of the MSCs. Parallel to the studies in humans, we propose to evaluate the

response of experimental autoimmune diabetes (a genetically determined model

and a chemically induced model) to the infusion of the human MSCs. The extent of

the pathogenic autoimmune response will be evaluated in these models, as well as

the role of the MSCs in the regeneration of the pancreatic tissue damaged by the

autoimmune aggression, during the early stage of the disease.

The results obtained from this project will demonstrate the therapeutic

effect of the infusion of MSCs in autoimmune diabetes treatment in humans and

will also elucidate the immune and regenerative mechanisms of action involved in

this therapeutic response through the studies in animal models presenting

autoimmune diabetes. If a good quality therapeutic efficacy is demonstrated, the

infusion of MSCs could become a therapeutic alternative for the treatment of DM-1,

which is an autoimmune disease associated with a low quality of life and requires

chronic treatment.

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General Objective

This project has, as a general goal, to evaluate the safety, the therapeutic

efficacy and the mechanisms of action of the human mesenchymal stem cells

infusion for the treatment of recently diagnosed type 1 diabetes mellitus. We have

established the following specific goals:

Methodology

Fourteen patients having recently diagnosed DM-1 (for less than 4 weeks;

antibody anti-GAD65 positive) will be receive an infusion of allogenic MSCs at

Unidade de Transplante de Medula Óssea do Hospital das Clínicas da FMRP-USP.

This research project and its informed consent form have been approved at Local

and National Ethical Committeee for Humans Research. The bone marrow donor

(BM) will be a healthy member of the family of the diabetic patient selected on the

basis of HLA typing. Preferentially a donor who does not present diabetes-related

HLA (DRB1*03 and/or *04 and DQB1*0201 and/or *0302) will be used. The donor of

the BM will be submitted to the collection on iliac crest of, approximately, 100 ml

of bone marrow (BM). The BM samples from the donors will be processed at

Laboratório de Terapia Celular do Centro de Terapia Celular (CEPID-FAPESP) of

Centro Regional de Hemoterapia do HC-FMRP-USP. The MSCs will be cultivated in

sterile conditions within the strict requirements of good manufacturing practice

(GMP) for the production of cellular components for infusion in humans. Samples of

BM of healthy individuals (volunteers BM donors) will be collected for the isolation

of MSCs for infusion in animal models of diabetes. The PBMCs will be isolated from

BM by density gradient centrifugation (Ficoll HypaqueTM, d=1,077), then 4x107 BM

mononuclear cells will be ressuspended in α-MEM medium supplemented with 15%

of fetal bovine serum (FBS) and cultured at 37ºC in 5% CO2.

After three days, the non-adherent cells will be removed, and 50 ml of

medium will be added. The medium will be changed every 3-4 days and when the

cells are semi confluent, they will be dissociated using tripsin and expanded (first

passage). The MSCs will be submitted to successive passages (3-5) until a sufficient

number of cells are acquired for the procedure (1-2 x106/kg of the patient in each

infusion). Three days before the infusion of the MSCs in the patient, the FBS

(medium) will be replaced by autologous serum (at 5-10%). A second infusion of

MSCs will be carried out, both with the same number of cells (1-2 x106/kg) and

separated by 30 days. Samples of the MSCs will be separated before the infusion

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for: 1) morphologic, immunophenotypic and cytogenetic characterization; 2)

adipogenic and osteocytic differentiation; 3) evaluation of the in vitro

immunosuppressant capacity. After the infusion of the MSCs, the patient will be

monitored weekly until D+60 post-infusion and, then, every 3 months. The

monitoring will be done by clinical and laboratory exams (which include glycemic

fasting, C-peptide and glycosated hemoglobin levels). Peripheral blood of the

patients will be collected for the study of the immune response on days +1,+2, +7,

+14, +30, +60, +180, +270, +360 and, then, every 6 months after the second

infusion of the MSCs.

Focusing on the study of the regenerative and immune systems of the action

of infusion of human MSCs (isolated from BM) for the treatment of experimental

autoimmune diabetes, we will use a genetically determined model of diabetes

(NOD mice, Non-Obese diabetic mouse) and a chemically induced autoimmune

model of diabetes induced by administering streptozotocin (STZ) in mice C57BL/6.

The C57BL/6 mice (male, 8 weeks old) will be submitted to 5 consecutive daily

doses of STZ. Five days after the last dose of STZ (initial phase of the disease) the

C57BL/6 mice will endogenously receive the infusion of human MSCs. The NOD

female mice, 12 weeks old (initial phase of the disease) will be infused with human

MSCs. After the infusion of the MSCs, the mice will be monitored twice a week in

relation to glycemia and weight. After 10, 17, 24, 32 and 60 days from the

infusion, the animals will be sacrificed and the pancreas, spleen and serum of

these animals will be collected for immunologic analysis. We will evaluate the

occurrence of fusion between human MSC cells and pancreatic murine β cells

through FISH analysis of X and Y chromosome for murine and human.

Immunohistochemistry will be performed to evaluate the capacity of human MSC

cells to differentiate into insulin-producing β cells. In addition, MSCs will be

infected with recombinant retrovirus that carries the LacZ gene for studies of

migration and stability of MSCs in the injured tissue.

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Subproject 29: Treatment of multiple sclerosis with hematopoietic stem cells:

evaluation of the clinical response and immune mechanisms of action

Abstract

In the past years, clinical studies have demonstrated that high doses

immunossuppression (HDI), followed by the autologous transplant of hematopoietic

stem cells (AHSCT) is able to suppress the inflammatory activity in patients who

have autoimmune diseases (AID) and can induce long clinical remissions. The AHSCT

in AIDs is based on the idea that intense immune ablation may eliminate the auto-

reactive cells. The immune system reconstituted from hematopoietic precursors

may reestablish immune tolerance. Over 1,000 transplants in clinical trials phases

I/II have already been performed. Recently, randomized phase III clinical trials

comparing the AHSCT with conventional cell therapy for AIDs diseases, were

initiated in the United States and Europe.

In Brazil, the treatment of AID with HDI followed by AHSCT, has began at the

Bone Marrow Transplant Unit in the Hospital das Clínicas da Faculdade de Medicina

in Ribeirão Preto – Universidade de São Paulo(USP) in September 2001, it focused

on severe rheumatic (Systemic lupus erythematosus and multiple sclerosis {MS})

and neurologic diseases (secondary progressive multiple sclerosis), which do not

respond to conventional treatments. HDI therapy, followed by AHSCT led to the

remission of the disease in 75% of the MS patients (secondary or primary

progressive forms) transplanted at hospitals which participate in the multicenter

study (N=41), confirming the in the literature. In these patients, the value of EDSS

(Expanded Disability Status Score) improved or stabilized after transplant.

In addition to clinical studies our group has studied the action mechanisms of

HDI/AHSCT for the treatment of organ-specific AIDs, i.e., which are the induced

changes in the immune system of the patient caused by HDI/AHSCT could explain

the remission of the AID. Our results and recent reports in literature, suggest that

the HDI/AHSCT induce a “reprogramming” of the immune system after the AHSCT,

i.e., a regeneration of a “new” or “different” and self tolerance in patients with

AIDs. However, additional molecular and cellular immune studies are necessary and

important to better understand the mechanisms of action of HDI/AHSCT for the

treatment of (Type-1 diabetes mellitus) DM-1 and MS. The knowledge of these


82

diseases remission mechanisms is fundamental for the establishment of the

HDI/AHSCT therapy as an alternative way for the MS and DM-1.

The success of these first experimental clinical protocols of phase I/II has

opened perspectives to use the HDI/AHSCT therapy in other groups of patients.

Thus, this project will evaluate if the AHSCT preceded by the HDI is able to induce

long clinical remission in patients who have remissive MS (inflammatory, recurrent

to the treatment with interferon), as well as investigate the molecular mechanisms

of the therapeutic action of the HDI/AHSCT in this disease.

General Objective

This project has as a general objective, to determine if the autologous

transplant of hematopoietic stem cells, preceded by immunossuppression in high

doses is able to induce long clinical remission in patients who have relapsing-

remitting MS, as well as investigate the molecular and immune mechanisms of the

therapeutic action of the HDI/AHSCT in these diseases.

Methodology

Patients with MS relapsing-remitting (N=15) will be submitted to the

treatment with HDI/AHSCT at the Bone Marrow Transplant Unit in the Hospital das

Clínicas da Faculdade de Medicina in Ribeirão Preto – USP. The will be monitored,

after the transplant, in regards to clinical and laboratorial response. The clinical

project of the HDI/AHSCT for the treatment of MS was approved by the Ethical

Committee of the Hospital das Clínicas of FMRP-USP (Proc. 15503/05) and by the

National Ethical Comitte for Research (25000.056576/2006-56; RG 12908). This

clinical project is part of a phase III clinical trial, multicenter and randomized

(MIST, Multiple Sclerosis International Stem Cell Transplant Trial) to evaluate the

efficacy of the HDI/AHSCT in comparison with the conventional treatment

(Interferon, Copaxone ou Mitoxantrone). Other participant centers of the MIST are:

Northwestern University (Chicago, USA), University of California (San Diego-CA,

USA), Baylor College of Medicine (Houston-TX, USA), Charite Hospital (Berlin,

Germany), Singapore General Hospital (Singapore) and Hospital Israelita Albert

Einstein (São Paulo-SP, Brazil).

Peripheral blood samples will be prospectively collected from patients who

had MS relapsing-remitting before (pre-mobilization) and after the transplant

83

(D+180, D+360, D+540, D+720), for isolation of the T CD4+ e CD8+ cells. The gene

expression profile of the T CD4+ e CD8+ cells will be compared between: 1)

pre/post-AHSCT patients and healthy individuals; 2) pre-AHSCT and post-AHSCT

patients. The analysis of the gene expression will be done using the cDNA

microarrays technique (platform Agilent, EUA), that is already standardized in the

Hematolgy Laboratory- Centro de Terapia Celular (CEPID FAPESP) do Centro

Regional de Hemoterapia do HC-FMRP-USP. The results of the gene expression

profile studies will be analyzed by bioinformatics programs which are specific for

the determination of differentially expressed genes. The results will be validated

by the RT-PCR technique, using 7500 Real Time PCR System (Applied Biosystems),

which is well standardized at our laboratory.

Subproject 30: Mesenchymal stem cell (MSC) for treatment and prevention of

graft versus host disease (GVHD) in patients transplanted with hematopoietic

stem cells

Abstract

The allogenic transplantation of hematopoietic stem cells is considered to be

the therapy of choice for the treatment of several diseases, among them

hematological neoplasm and other hematological diseases. However, despite the

immune suppression of the patients they may trigger a series of secondary effects,

such as graft-versus-host disease (GVHD). GVHD is the main cause of morbidity and

mortality in patients submitted to bone marrow transplant (BMT). This disease is

due to the activation of T cells in response to the allogenic MHC after bone marrow

transplantation. The T lymphocytes of the donor identify the antigens of the host

as foreign bodies and trigger the immune response against them. The GVHD is

normally classified as acute (generally occurring up to 100 days after the

transplant, with skin, intestine and liver involvement) or chronic (generally

occurring over 100 days after the transplant, characterized by lesions, dryness,

constriction or several organs sclerosis).

The mesenchymal stem cells (MSC) are rare and are present in the bone

marrow in a frequency of 0,001% to 0,01% of all nucleated medullar cells and can

be identified by the positivity of four markers: Stro-1+, CD73 (SH3/SH4), CD105


84

(SH2) and CD90 (Thy-1) and negativity for endothelial (CD31, KDR, VE-cadherin)

and hematopoetic cells markers (CD34, CD14 and CD45). These cells adhere to

plastic and can be cultivated in the presence of fetal bovine serum (FBS), with

rapid multiplication of approximately 40 fold. Therefore, about one billion

mesenchymal cells are obtained from the culture of one single cell. MSCs express

intermediate levels of MHC class I, and do not express the MHC class II antigens on

their surface. The expression of MHC class I is important and prevents the MSCs

from being eliminated by the NK (natural killer) cells. On the other hand, the lack

of MHC class II and of co-stimulatory molecules (CD40, CD40L, CD80 and CD86)

permit to this cell to develop immune vigilance escape mechanisms, preventing the

activation of T CD4+ and T-CD8+ lymphocytes. This immunossuppressor potential

led several researchers to develop clinical studies using MSCs for the treatment of

GVHD and, in many cases, successful results have been reported. Similarly, the

present project aims to use MSCs for the treatment and prevention of the graft-

versus-host disease in patients submitted to the hematopoietic stem cells

transplant.

General Objectives:

To contribute to the formation of human resources, nucleation of the staff

and integration with multidisciplinary teams to develop clinical protocols for cell

therapy.

To determine the efficiency and safety in the use of allogeneic mesenchymal

stem cells expanded in vitro for the treatment of patients who developed the

graft-versus-host disease after allogeneic hematopoietic stem cells transplantation.

Methodology:

Isolation, selection and cell culture of MSCs from blood and the bone

marrow. The PBMCs will be isolated by density gradient centrifugation (Ficoll

HypaqueTM, d=1,077), followed by cell number counting in Neubauer chamber for

further cell plating. After 72 hours of culture, non adherent cells will be removed

and the attached MSCs will be expanded in culture.

Determination of the number of colony forming unit (CFU). Mononuclear

cells will be cultured on Petri dishes and the number of colonies counted from day

5 to 7.

85

Immunophenotypic Characterization. A panel of 18 monoclonal antibodies will be

used: CD105-FITC (SH-2) BD; CD73-PE (SH3/SH4) Pharmigen; Stro-1 supernatant

fluid ascite; CD90-PE (Thy-1) BD; CD29-PE (integrin b-1) BD; CD13-PE

(aminopeptidase-N) BD; HLA ABC-PE Pharmigen; CD49e-PE (VLA-5) BD; CD54-PE

(ICAM-1) BD; CD44-FITC (HCAM-1) BD; CD51/61-FITC (Avb5) Pharmigen; CD106-PE

(VCAM-1) BD; CD34-PE (Sialomucyn) BD; CD14-PE (LPS-R) BD; CD-45-FITC (LCA) BD;

HLA-DR BD; CD31-FITC (PECAM-1) BD; KDR (VEGFR-2) Sigma e Isotype Simultest

(IgG1/IgG2) BD. Cells (5x105 cells) are analyzed after incubation with antibodies by

flow cytometry as recommended by the manufacturer.

Differentiation potential: The adipogenic, osteogenic and chondrogenic

differentiation potentials will be evaluated as previously described (Covas et al.,

2003)

Cytogenetic profile: Samples from bioreactors and static cultures in

exponential growth will be subjected to treatment with colcemid (0.15 mg/mL) for

4 hours at 37°C, followed by incubation with KCl (0.075M) for 20 minutes.

Subsequently, the cells will be fixed with methanol and acetic acid, washed, and

transferred to slides that will be kept at room temperature for a period of 24h

before proceeding with the trypsin digestion. The remainder of the material will be

stored in a freezer (-20°C) for the in situ fluorescence hybridization techniques

(SKY and FISH). The chromosomal banding technique will be performed by

incubation with a trypsin solution (1%), followed by washing in tap water and

staining with Giemsa for 5 minutes. The slides will be ready for analysis after the

staining.

86

INDICATORS

The following indicators are numerical indicators for our estimates of the

production of the Project for the next five years (2009-2013).

Bibliographic production

Number of published papers specialized peer reviewed journals: 45

Number of thesis concluded (Master/Doctorate): 40

Number of deposited patents : 6

Number of substances or new articles propagated in the media printed or

electronics

Other products

Number of courses offered:

‐ Courses of Post Graduation “lato sensu” for Professors of biology area: 2

• Post Graduation Courses already offered: 9

• Post Graduation Courses to be created : 12

Number of participating students:

• Pos Graduate: 120

• Graduation (Scientific Initiation): 12

• Junior and high school students: 36.000

Number of hospital patients participating in the clinical studies: 60

Number of new developed animal models : 9

Number of derived cell lines: 20

Number of constructed vectors: 15

Number of concluded projects: 30

Number of expositions or presentations: 12

Number of accesses to the homepage of the INCTC: >20.000

87

D) Program for High Qualified Human Resources Formation

The proposal of this project aims in special at the formation of highly

qualified human resources focused on capacity of interacting in all levels of Cell

therapy process, since the generation of cells with the necessary characteristics up

to the clinical studies. The complex methodologies employed in this project is a

great opportunity for post graduation students and researchers to interact by

sharing experiences through several laboratories of the Institutes and also with

international collaborators.

Running Courses

COURSE INSTITUTE CODE HOURS CREDITS PLACESTopics in Cell Culture for Cancer Research

FMRP/USP/Ribeirão Preto

RCM5816 60 hours 4 9

Molecular Methods applied to Hemotherapy

UNESP/Araraquara 75 hours 5 10

Cellular and Molecular Mechanisms Related to the Physiopathology of Leukemias

FCF/USP/ Ribeirão Preto

6046009-1 75 hours 5 10

Immunotherapy: Fundamentals and Clinical Applications


RIM5754 30 hours 2 10

Cellular Therapy in Inflammatory, Autoimmune and Neoplasics Diseases: Basic and Clinical Aspects


RCM5813 30 hours 2 10

Stem cells and Animal Cloning

FMVZ/USP/São Paulo VCI5779 60 hours 4 20

Placenta Morphophysiology and Placentation of and


Biology of Development


88

NEW COURSES

COURSE INSTITUTE HOURS PLACESPost Graduation Course latu senso in Biology – UNIVESP

Plataform for distant studies

360 hours 900 teachers36.000 students

Post Graduation Course latu senso in Biology - Pro-Reitoria de Cultura e Extensão

Plataform for distant studies

360 hours 40

Advances in Molecular Methods for Investigation in Biology

FCF/USP/ Ribeirão Preto

75 hours 15

Use of animal models for leukemia studies.


60 hours 6

Concepts in Biotechnology and Medical Field Applications


60 hours 15

Interdisciplinarity: A Tool for Socialization Knowledge and Citizenship Construction

Museums and Sciences Spaces

45 hours 40

Use of Museums and Sciences Centers Resources for Learning Dynamization

Museums and Sciences Spaces

60 hours 40

Language, Writing and Scientific Diffusion

EAD platformMoodle

45 hours 40

Scientific Diffusion Workshop EAD platform Moodle 30 hours 40Research Methodology as Diffusion Tools

EAD platform Moodle 60 hours 40

Midia and Scientific Diffusion EAD platform Moodle 30 hours 40Mediation in Centers and Sciences Museums

EAD platform Moodle 30 hours 40

Fundamentals in Scientific Education EAD platform Moodle 30 hours 40

Human Resources formation by the Coordinators

Supervisor Student Year of concludion Actual position Institute Country

Roberto Passetto Falcão

Júlio César Voltarelli 1981

Titular Professor, Coordinator of Transplant Unit of HCRP-USP

Department of Clínica Médica, Faculty of Medicine of Ribeirão Preto - USP

Brazil


Eduardo Antonio Donadi

1986 Associate Professor


Brazil


Silvia Beatriz Vieira Ramos 1994

Assistant Professor of Obstetrics & Gynecology

Division of Reproductive Endocrinology & Infertility University of North Caroline

Chapel Hill


Gil Cunha de Sanctis 1993 Medical Director

Fundação Hemocentro de Ribeirão Preto

Brazil


Raul Antonio de Moraes 1995

Adjunct Professor Research Coordinator of Fundação HEMOPE de Pernambuco

Department of Clínica Médica, Faculty of Medicine of Pernambuco University

Brazil

89



Eduardo Magalhães Rego



Brazil


Sergio Luiz Martins 2001

Clinical Analysis Division Coordinator

Laboratório Fleury Brazil


Cintia Machado 2002

Adjunct Professor Coordinator of Flow Citometry Laboratory of Fundação Hemope de Pernambuco

Department of Clínica Médica, Faculty of Medicine of Pernambuco University

Brazil


Rodrigo Tocantins Calado

2003

Hematology Branch, National Heart, Lung, and Blood Institute

National Institutes of Health EUA


Edgar Gil Rizzatti 2005

Haematology Section Coordinator

Laboratório Fleury Brazil


Nilce Marzola Ideriha 1982 Associate

Professor

Faculty of Medicine of Universidade Estadual de Londrina

Brazil


Suzana Beatriz Veríssimo Melo 1982 Associate

Professor Faculdade de Medicina da USP-SP Brazil


Aureo Evangelista Santana


Faculty of Veterinary Medicine of Jaboticabal, UNESP

Brazil


José Orivaldo Mengele 1993 Titular Professor

Centro de Pesquisas Gonçalo Moniz – Osvaldo Cruz Foundation Fiocruz Salvador, Bahia

Brazil

Lewis Joel Greene

Augusto Cesar Cropanese Spadaro

1977 Titular Professor

Department of Physics and Chemistry School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo

Director

Lewis Joel Greene

Leila Maria Beltramini Sabbag


Instituto de Física, Departamento de Física e Informática, São Carlos, SP , Universidade de São Paulo

Brazil

Lewis Joel Greene

Silvia Nassif Del Lama 1982 Adjunct

Professor,

Department of Genetic and Evolution, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, SP.

Brazil

90


Lewis Joel Greene

Lusiane Maria Bendhack 1984 Associate

Professor


Brazil

Lewis Joel Greene Clarice Izumi 1985 Laboratory

Specialist

Centro de Química de Proteínas, Faculty of Medicine de Ribeirão Preto, Universidade de São Paulo

Brazil

Lewis Joel Greene

Vitor Marcel Faça 1998 Pos doctoral

Fred Hutchinson Cancer Research Center, Seattle

USA

Lewis Joel Greene

Sandra Pereira Rodrigues 2000 Pos doctoral

Fred Hutchinson Cancer Research Center, Seattle

USA

Lewis Joel Greene

Gustavo Antônio de Souza

2002 Professor

The Gade InstituteSection for Microbiology and Immunology , University of Bergen,

Norway

Lewis Joel Greene

Fabíola Leslie A. C. Mestriner

2006 Laboratory Specialist

Department of Farmacology, Faculty of Medicine of Ribeirão Preto, SP, Universidade de São Paulo

Brazil

Lewis Joel Greene

Augusto Cesar Cropanese Spadaro

1979

Titular Professor titular Director of School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo


Brazil

Lewis Joel Greene

Almir de Souza Martins 1996 Adjunct Professor

Department of Phyisiology and Biophysics, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG.

Brazil

Lewis Joel Greene

Julio César Padovan 1996 Researcher Rockefeller

University

New York, USA

Lewis Joel Greene

José César Rosa 1998 Professor

Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos, Faculty of Medicine of Ribeirão Preto, Universidade de São Paulo.

Brazil

91


Lewis Joel Greene

Carolina Bosch Cabral 2004 Pos doctorate

Massachusetts General Hospital , Harvard Medical School, HMS,

USA

Lewis Joel Greene

Lyris Martins Franco de Godoy

2004 Pos doctorate

Max Plank Institute of Biochemistry, Department of Proteomics and Signal transduction

Germany

Lewis Joel Greene

Maria Sumiko Arita Matsuura

professor retired

Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP.

Brazil

Lewis Joel Greene

José Godinho Neto 1991 a 1993 Assistant

Professor

Departamento de Química, Universidade Federal Rural do Rio de Janeiro

Brazil

Dimas Tadeu Covas

Ana Cristina Silva Pinto 2007 Pos doctoral,

Medical Doctor

Unité Mixte du Recherch UMR 763 (INSERM) - Hospital Robert Debre - Paris

France

Dimas Tadeu Covas

Karen de Lima Prata 2006 Medical Doctor


Brazil

Dimas Tadeu Covas

Ana Paula Costa Nunes da Cunha Cozac

2004 Medical Doctor Fundação Hemocentro de Ribeirão Preto

Brazil

Dimas Tadeu Covas

Maria Ângela Pignata Ottoboni

2004 Supervisor

Laboratory of Quality Control, Fundação Hemocentro de Ribeirão Preto

Brazil

Dimas Tadeu Covas

Eugenia Maria Amorim Ubiali 2003 Medical

coordinator


Brazil

Dimas Tadeu Covas

Marina A Coutinho 2003 Medical Doctor Laboratorio Dr

Coutinho Brazil

Dimas Tadeu Covas

Benedito de Pina Almeida Prado Jr

2002

Medical Doctor, Head of Transfusional Agency

Unidade de Emergência – HCFMRP-USP

Brazil

Dimas Tadeu Covas

Luiz Paulo Cicogna Faggioni

2001 Medical Doctor Agência Nacional de Saúde Suplementar Brazil

Dimas Tadeu Covas

Simone Kashima Haddad

1996 Supervisor

Molecular Biology Laboratory, Fundação Hemocentro de Ribeirão Preto

Brazil

Dimas Tadeu Covas

Virgínia Proença Picanço Castro

2006 Pos doctoral Fundação Hemocentro de Ribeirão Preto

Brazil

Dimas Tadeu Covas

Silvana Maria Quintana 1997 Professor

Ginecology and Obstetrics Department-FMRP-USP

Brazil

92


Dimas Tadeu Covas

Elisa Maria Carbolante 2004 Professor

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, SP, Universidade de São Paulo

Brazil

Dimas Tadeu Covas

Antonio Adilton O Carneiro

2003 Professor Faculdade Filosofia Ciências e Letras de Ribeirão Preto – USP

Brazil

Dimas Tadeu Covas

Evamberto Garcia Góes 2001 PIPE fellowship FAPESP Brazil

Dimas Tadeu Covas

Rita de Cássia Viu Carrara 2001 Pos Doctoral


Brazil


Rodrigo Siqueira Abreu e Lima

2007 Head Oncology Center of Brasília Brazil


Gil Cunha de Santis 2006 Medical Director

Fundação Hemocentro de Ribierão Preto

Brazil


Lorena Lobo Figueiredo Pontes

2008 Doctorate

Hospital das Clínicas da Faculdade de Medicina de Ribierão Preto

Brazil

Julio César Voltarelli

Márcia Piuvesan 1997 Pos doctoral Universidade Federal

da Paraíba Brazil


José Hermênio C. Lima Filho 1998 Associate

Professor Universidade Federal do Paraná Brazil


Maria Angélica Watanabe 1998 Associate

Professor Universidade Estadual de Londrina Brazil


Ana Beatriz Pereira Lima Stracieri

1999 Medical doctor Hospital das Clínicas FMRP-USP Brazil


Fabíola Attiê de Castro 2001 Associate

Professor

Faculdade de Ciências Farmacêuticas de Ribeirão Preto- USP

Brazil


Kelen Cristina Malmegrim 2006 Pos-doctoral Hemocentro de

Ribeirão Preto Brazil


Luís Paulo Faggioni 2006 Medical doctor Agência Nacional de

Saúde Brazil

Julio César Voltarelli Sulani S. Souza 1996 Medical doctor Universidade Federal

de Brasília Brazil


Márcia Cristina Furtado Nascimento

1999 Medical doctor Instituto Oswaldo Cruz, Salvador-BA Brazil


Marta Maria L. Lemos 2000 Medical doctor Hospital AC

Camargo-SP Brazil

Julio César Voltarelli Rejane Vieira 2001 Medical doctor Hospital Geral de

Fortaleza-CE Brazil


Eduardo AJ Paton 2003 Medical doctor,

director Hospital do Câncer, Barretos-SP Brazil


Maria Carolina O. Rodrigues 2006 Medical doctor Hospital das Clínicas-

FMRP-USP Brazil


Vivian Youssef Khouri- 2007 Dentist

Hospital das Clínicas-FMRP-USP

Brazil

93


Maria Angelica Miglino

Maria Rita Fernandes Marchado

1995 Assistant Professor

Departamento de Morfologia e Fisiologia, FCAV/UNESP Jaboticabal

Brazil


Francisco Solano Feitosa 1997

Professor of Veterinary Surgery

Universidade Federal do Piauí Brazil


Antonio Augusto Coppi Maciel Ribeiro

1998 Livre Docente Professor in Animal Anatomy

FMVZ/USP Brazil


Cesar Augusto Water 1999

Professor Doctor in Animal Reproduction

Universidade Estadual do Ceará Brazil


Francisco de Sales Rezende Carvalho

1999 Professor Doctor in Equine Surgery

Universidade Federal de Uberlandia Brazil


Fernando Antônio Ferreira

1999

Professor in Veterinary Clinical and Surgery

Universidade Federal de Uberlandia Brazil


Adelmar A.Amorim Junior

2000 Professor Doctor in Anatomy

Universidade Federal do Recife Brazil


Miguel Ferreira Cavalcante Filho

2000 Professor Doctor in Animal Anatomy

Universidade Federal do Piaui Brazil


Rosa Helena dos Santos Ferraz


Universidade Federal do Mato Grosso Brazil


Carlos Rosemberg Luis


Universidade Federal do Goias Brazil


Rosana Marques e Silva

2001 Professor in Animal Anatomy

Universidade de Brasilia Brazil


Luciana Silveira Flores Schoenau


Universidade Federal de Santa Maria - RS Brazil


Williams Gomes Neves 2001

Professor Doctor in Animal Anatomy

Universidade Federal do Piaui Brazil


Marilyne José Afonso A. Lins Amorim


Universidade Federal do Pernambuco Brazil


Maria Amélia Zogno 2002 Technician

Graduate Level FMVZ/USP Brazil


Tatiana Carlesso dos Santos


Universidade Estadual de Maringá Brazil


Moacir Franco de Oliveira 2004

Professor Doctor in Animal Anatomy and assistant Head of the Rector

UFERSA Universidade do Semi-árido Nordestino

Brazil

94



Flávia Thomaz Verechia Pereira

2004 Professor Doctor in Histology

UNESP – Dracena Brazil


Carlos Eduardo Ambrosio 2004

Assistance Professor in Animal Anatomy

FMVZ/ USP Brazil


Antonio Chaves de Assis Neto


UNESP/Dracena Brazil


Lourivaldo Paz Landim 2005 Technician

Empresa de Biotecnologia da Reprodução, BIOEMBRYO - Fertilização in vitro.

Brazil


Daniele dos Santos Martins 2006 Researcher

Instituto Butantan Brazil

Maria Angelica Miglino Erika Branco 2007

Professor Doctor in Animal Anatomy

Universidade Federal Rural da Amazonia Brazil

95

E) Detailed Description of the Actions for the Transfer of Knowledge to Society

Science is a powerful enterprise that can substantially improve the life of

people. Scientists have had and will continue to have a fundamental participation

in the development of new medications and treatments that affect millions of

persons and of new techniques that have improved the quality of life, such as the

modern systems of communication and the computers, as well as the development

of new technologies that will permit the sustainable development of civilization on

this planet of limited resources. All of these aspects are and will continue to be

fundamental for the survival of man, so that the development of an ample

scientific culture is indispensable in order to guarantee a better future for the next

generations. The maintenance and growth of this scientific culture requires training

not only of professional scientists, but also the scientific education of a wide

segment of society, including engineers, journalists, lawyers, health professionals,

and mainly science teachers who, together with their students at the elementary

and high school level, are at the basis of this immense system.

In addition, scientific reasoning provides individuals with a secure basis for

the development of language, of logic and of the ability to solve problems in the

most varied and extensive fields of human activity, causing scientific education to

be a necessary endeavor in order to face the new requirements of the 21st century.

The objective of the present project is to reduce tne time between the

production of knowledge and the acquisition of knowledge by society. In today’s

world, various spaces can be used for informal education, science education, and

the dissemination of the knowledge produced. The various professionals involved in

the INC&T intend to consolidate within a period of 36 months a program of informal

science education with an ample capacity to train human resources (HR). During

the first 12 months, the authors of the scientific projects will contribute topics of

interest to society or to an information bank for the purpose of science education.

The experiences originating from projects of informal education and dissemination

of the knowledge of the team will form another data bank, which will be

incorporated into the first. Between the 13th and 24th month, individuals able to

produce materials, products or other mechanisms for the dissemination of

knowledge on the basis of catalogued experiences associated with the available

96

knowledge will be identified among the HR of the IN. A lato and stricto sensu

Postgraduate course in the dissemination of knowledge and in informal scientific

education will be implemented, with 70% of the course load (CL) offered at a

distance (distance education, DE) and 30% in the presence of the students. The

latter CL will occur in spaces of scientific dissemination of the partner institutions

or of the community and the CL at a distance will be structured in an ADE Program

monitored by the Moodle Platform. Inviduals with the ability to disseminate science

will be identified among the specialists and assigned to a group that will start a

class of professionalizing master’s education. Between the 24th and 36th month, the

IN will assess the activities of informal education with emphasis on visits to

museums in order to make available the validated experience to society. The

research of the Master’s students will have to focus on scientific topics of social

interest for the dissemination of knowledge or for the production of teaching

material, publications in the digital media or in print, and science kits among other

possibilities. During the last six months the partners will assess the various phases

of the process of construction of the group of specialists and master’s graduates on

the basis of group experience, in order to identify in each HR the ability to include

young people in the actions and to create products and competence for itinerant

education, among others. The experiences of the proposing team involve many

ongoing projects, the actions of five museums or science spaces, as well as the

participation of the investigators in numerous regional, national and international

experiences of HR training and science education, which will give sustainability to

this proposal.

This project of dissemination of knowledge will be the responsibility of the

following five partners: Casa da Ciência (House of Science) FMRP/USP,

Interdisciplinary Dynamic Museum (MUDI)/UEM, Museum of Human Anatomy

(MAH)/UnB, Museum of Comparative Anatomy Professor Plínio Matos Pessoa

(MAC)/FMVZ/USP, and Interdisciplinary Science Museum (MIC)/UNIPAR. Their

current activities permit the citizens to spend time in research environments in a

ludic and interactive manner. The House of Science carries out educational

projects with laboratory activities in schools, science fairs, in research groups and

in courses for teachers; it publishes a science bulletin and is involved in the the

innovative project called Talent Hunt. The collection of MAV FMVZ dates back to

1940 and the museum was opened to visitors in 1984, currently exhibiting the

97

largest comparative anatomy collection in Brazil, which is included in projects of

social inclusion, itinerant activities, and science education. The MAH was founded

in the 1970’s and was opened to visitors in the 1980’s. It is part of the tourist

itinerary of the federal capital and has maintained since then an ample project of

continued action in science education and in the support of science fairs in Brasília

and surrounding area. The MUDI has 23 yars of experience in the training of human

resources (elementary and high school teachers) and its team is currently involved

in more than 30 projects of dissemination, participating in interactive exhibits and

science fairs, with extensive experience in itinerant activities. The patrimony of

the four museums involved has a great potential for the dissemination of

knowledge and for scientific initiation.

GENERAL OBJECTIVES

To establish an information network among researchers that will permit

them and the team of scientific dissemination of INC&T to discuss what part of

research would be interesting to disseminate and when and how.

To set up a well-qualified program of HR training in order to disseminate

knowledge.

98

SPECIFIC OBJECTIVES

To produce a data and/or scientific information bank covering various topics

linked to the projects of the National C&t Institute for purposes of informal science

education focusing on Brazilian children and adolescents and the population in

general.

To identify among peers initiatives already existing for the dissemination of

information and to catalogue them by topic, type and specialty in order to use

them during the execution of the mission of the Institute.

To encourage young investigators to participate in projects or initiatives of

dissemination of knowledge involving high school and university students.

To identify human resources with the skill of translating the scientific language in

order to disseminate and popularize the knowledge produced.

To train human resources in postgraduate courses of Distance Education (DE)

using the Moodle platform. To prepare teams with competence to receive scientific

information and to identify ways of organizing it for dissemination, with emphasis

on elementary education according to the mission of CAPES.

To train HR with the skill and competence needed to elaborate different

modalities (actions) of dissemination of knowledge based on trans-disciplinarity and

on the educational level of the target public to be reached.

To format actions of dissemination that will reach large groups of people.

To have itinerancy as the guiding principle of the actions of dissemination

and popularization of the knowledge produced.

To select international, national and regional events for the dissemination of

the knowledge produced such as National C&T Week, National Museum Week, IES

Extension Weeks, Science Fairs, Agriculture and Animal Husbandry Fairs etc.

The project of dissemination of knowledge and science education should

reach the goals established within three years as follows:

Goal 1: duration, 12 months

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Stage 1: To organize a data bank compiled by the investigators of IN and of

the partner museums in order to disseminate the knowledge produced in an

interdisciplinary manner.

Stage 2: To identify among partners the experiences in dissemination in

which they participated, their forms and their results in order to set up a data bank

consisting of the experiences of the group, with replication of viable procedures at

the national level.

Goal 2: duration, 18 months

Stage 1: To produce material for dissemination generated from scientific

data banks and from experiences of dissemination, catalogued in Stages 1 and 2 of

Goal 1, with the addition of current literature for the construction of dissemination

materials obtained in this phase. E.g.: science kits, sites, informative posters,

newspaper and magazine articles, interviews, identification of lecturers, courses,

science workshops, thematic exhibits, CDs, DVDs, films etc.

Stage 2: To implement lato and stricto sensu postgraduate courses:

specialization in the Dissemination and Popularization of Knowledge, with the

following characteristics: 18 months duration, hour load of 360 hours, 24 credits (1

credit = 15 hours of programmed activities; conclusion of the course in the form of

activity of scientific dissemination). Master’s degree: 24 months duration; hour

load of 450 hours; 30 credits and a thesis presented in the form of a method,

process or product of dissemination of knowledge, which will correspond to an hour

load equivalent to 15 credits. The courses will involve activities at a distance (70%)

and in the presence of students and teachers (30%).

Goal 3: 24 months duration

Stage 1: 24 months duration

To give continuity to the Interinstitutional Postgraduate course Stricto Sensu

(professionalizing Master’s degree) in scientific dissemination. A seminar of

scientific orientation will be held during this phase for definition and discussionof

the research topics between advisers and students.


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To analyze the information of the data bank as a whole in order to format

actions of scientific dissemination and popularization of knowledge.

To identify HR with the skill and competence for the transfer of knowledge,

production of materials for dissemination; ability to analyze the scientific discourse

for communication with the lay public; setting up interactive exhibits, fairs and

events; ability to produce scientific illustration, in addition to didactic and

informative texts.


To prepare a workshop in order to evaluate the activities of dissemination of

knowledge catalogued in the information and research data bank. To make

available to postgraduate students and recipients of fellowships the literature

acquired in the project, permitting them to mix concepts having interdisciplinarity

as a guide of the work for the conclusion of the course in formatting to be

presented at the workshop.

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F) Actions detailing for transfer of knowledge for the Business Sector

or for the Public Policy formation

In the past decades the global economy has fundamentally changed their

characteristics. Previously based mainly on land, labor and capital, the global

economy is based on current knowledge. The production and modern innovation

systems have as its main component the information and knowledge, reflecting a

society that has change deeply under the impact of the new technologies and it has

been properly called the Knowledge Society. In this new Knowledge Society the

public and private limits, between science and technology and between University

and Industry are gradually diminishing, causing a system overlapped that did not

exist before. In this scenario, it has appeared a new organizational context in

which industry, government and academia tend to integrate their interests and

goals. This new integrated environment University-Industry-Government was

considered as the double-helix of DNA, a triple-helix in development (Etzkowitz,

1996; 2000; Gebhardt, Pohlmann et al., 2004)

The CTC’s proposal for transferring technology and innovation is located in

this new context. We want to catalyze the development of a triple-helix which the

academic and research interests are fully integrated to the objectives of

government and the industry in line with modern times.

The transfer of technology for government or business sector is one of the

priorities of INCTC. The groups involved in this proposal have experience in this

area and the prospect is that these activities can be expanded.

The CTC-FAPESP supported the development of several projects which

resulted in effective incorporation of technology by the public sector. Two

examples illustrate this point:

a) - the development of a system for irradiation of blood and blood components

based on units of teletherapy installed in the hospital network(Chen, Covas et al.,

2001; Goes, Covas et al., 2004; Goes, Borges et al., 2006; Goes, Ottoboni et al.,

2008). The knowledge generated was made available for the entire network of

services and blood bank linked to the unified health system (SUS).

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b) - the development of a superconducting susceptometre (SQUID) for noninvasive

quantification of liver iron (Carneiro, 2003; Carneiro, Vilela et al., 2004; Carneiro,

Fernandes et al., 2005). A prototype of this equipment was built. It is operating in

the laboratory of medical physics at the Faculty of Philosophy, Science and Letters

of Ribeirao Preto - USP, and soon, new equipment will be installed in the clinic

dependencies of the Hemocentro of Ribeirao Preto.

Another important process and that will be expanded in INCTC; it is the

development of systems for heterologous proteins expression in human cells. The

dominant practice in the biotechnology industry is the production of recombinant

proteins in prokariotic systems or in non-human mammalian cells (CHO, BHK, etc.).

However, the proteins produced by these systems generally differ from the native

protein in terms of glycosylation and even in folding, although these proteins have

the same primary sequence. In an original and successful way, we developed a

system for efficient production of clotting factors VIII and IX of in human cell lines.

In the developed system, the factor VIII production, for example, was 15 to 20

times more efficient than the production in the CHO conventional system which

will allow a large gain in efficiency and economy. The developed system has been

patented through the Agency for Innovation USP. The scale-up process to the

industrial level has been done with funding by FINEP (Process 0927/07) and in

partnership with the IPT (Technology Institute of São Paulo) and with Plantarium

which is a private company. More recently, given the diversity of the group, we

started a study in Pirassununga to change the expression vector with a specific

promoter (b-lacto globulin) to use it in epithelial cells of bovine mammary gland.

The cells expressing the protein of interest will serve as the nucleus donor for the

animal production carrying this change.

In the sequence we want to develop other protein expression processes of

medical and biological interest, such as the clotting factor VII, growth factors such

as VEGF and cellular IGF, recombinant viral proteins for the generation of

diagnostic tests or to use it as antigens. Our competence in this area can be proven

by publications related to the theme (Fontes, Davis et al., 2006; Penteado,

Medeiros et al., 2006; Picanco, Heinz et al., 2007; Russo-Carbolante, Penteado et

al., 2007; Picanco-Castro, Fontes et al., 2008; Picanco-Castro, Russo-Carbolante et

al., 2008)

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The development of modified human cells will allow the large-scale

production of recombinant proteins and also it will allow the development of cell

therapy. As described in the subproject 22, for example, want to test the

treatment of hemophilic B mice through the liver transplant of modified stellate

(star-shaped) cells with the gene for clotting factor IX.

The development of these technologies is currently done in the research labs

of the CTC-Hemocentro. It was planned a construction of a new building of 5,000

m2 (Laboratory of Biotechnology) specifically for this purpose and with allocated

resources from the Health Department of the State of São Paulo.

Another subproject related to the development of biotechnological processes

is the scale-up production of stem-cells or progenitor cells in bioreactors. The goal

is to amplify the number of cells, without cell differentiation, with the purpose of

providing cells in sufficient numbers to the pre-clinical studies and clinical trials.

The subprojects 2 and 23 are supposed to develop this technology for the

cultivation of MSC and ESC respectively. These culture systems need to be

developed under GMP conditions (good manufacturing practices). In this particular

project, the institution CTC (Hemocentro de Ribeirão Preto) has a national

prominence position. It was the first place in Brazil to develop a quality system to

hemotherapy services based on the ISO standard. Hemocentro received the first

national certification in this area and the developed model is considered as

international standard.

All cell therapy services done by Hemocentro are certified by NBR ISO

9001:2000. This ensures an excellence standard similar or even greater than those

in many European or North American services. Moreover, the Hemocentro services

are also accredited by the American Association of Blood Banks (AABB) since 2003.

This whole experience in managing the quality of products and services was

used in the implementation of structures for the stem cell culture and to make it

available for cell therapy: the bank of umbilical cord blood and human stem cells,

the cryobiology sector and the human cell culture laboratories for therapeutic use

comprising four clean culture rooms equipped with HEPA filters and operating in

GMP conditions since 2006. This structure has allowed the realization of important

clinical work as one performed by Voltarelli et al. whose results were published in

JAMA (Voltarelli, Couri et al., 2007) .




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Other clinical studies of phase I are in progress, including the treatment of

DECHA post-transplant of bone marrow with mesenchymal stem cells. At the

moment this study has included 13 patients and the treatment of one type I

diabetes patients using the same type of cells. The next step in this project is to

obtain the AABB accreditation for cell therapy with stem cells, it should happen

next year.

All this developed technology (cell culture methods in GMP conditions,

quality control of products and processes, standard operating procedures,

qualification systems for products and suppliers, specific rules for cell therapy that

did not exist in this country before) are available to be transferred for both public

and private sector.

Moreover, the incubator companies linked to CTC-Hemocentro de Ribeirão

Preto intend to develop the next set of projects that will integrate the transfer

program of INCTC:

Programs and Projects of the Business Incubator

SUPERA will execute its actions and hold its events using of structured

programs that are already working or that will be set up. The entire program

content and the stages of execution have already been defined for all programs.

The projects are briefly summarized below.

a) Consultant ship and Capacitating Program: It will habilitate the

businessmen linked to SUPERA, permitting them to acquire knowledge in the

business area. It will support partnerships and will advise in the elaboration of

proposals for obtaining funds from financing agencies. The practices of this action

line involve:

Guidance for the elaboration of Business Plans; Analysis and Discussion of

Business Plans; Selection of Enterprises and/or companies and the signing of

contracts; Monitoring of companies and of entrepreneurs who are in the pre-

incubation phase by means of a monthly report and of an annual auditing; Provision

of specific consultant ships for the incubated companies covering all the areas of

management and of technological development, financial management, marketing

management, juridical consultant ship and norms; Periodic Updating of Business

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plans in the companies and elaboration of Business Plans by the entrepreneurs

attended in the “Project hotel” modality; Qualification of businessmen and

entrepreneurs by training for the development of business competence based on

administrative and technological knowledge; To subsidize the participation of the

members of the business incubator in business fairs with funds obtained from

SEBRAE and FINEP; Support for a successful move of resident businesses into the

market based on judicious evaluation of the Development Report and of the

Business Plans; Permanent qualification of the managers of the incubator;

Participation of the incubator in the Technological Park Project of Ribeirão Preto

also with the objective of supporting the creation of an environment favorable to

the permanence in the municipality of the businesses of technological basis that

graduate from SUPERA.

b) INCPAR Program – Incubators of Technological Basis in Partnership for the

Development of New Business: this is a project aiming at the prospection of new

projects and at the qualification of the companies incubated. This project, which is

financed by FINEP, is a partnership of 5 incubators with a technological basis in the

state of São Paulo: SUPERA of Ribeirão Preto (coordinator of the project), CIE of

São José do Rio Preto, INAGRO of Jaboticabal, IEJ of Jaboticabal, and the

Technological Incubator of Botucatu. The project acts on two main fronts:

• In the prospection of research projects or of technologies with a market

potential together with universities. In this respect, the presence of USP in

Ribeirão Preto and of UNESP in Botucatu, Jaboticabal and São José do Rio Preto

should be pointed out, in addition to several private universities. These public

universities have in common research linked to biotechnology and health. The focus

of this prospection will be in the areas of concentration of the investigations

developed by these universities which, along general lines, are: Agronomy,

Biological Sciences, Medical Equipment, Human Health, Animal Health, and

Information Technology. Although these areas are a priority, other areas can also

participate in the IncPAR.

• In actions aiming at the growth and strengthening of the businesses

incubated, such as: the offer of qualification courses and of specialized services

directed at increased competitiveness of these businesses and at the expansion of

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their markets, and financial support for the participation in business fairs (Site

www.incpar.com.br)

c) National Program of support to the qualification and prospection of

new biotechnology projects (Competition for the BioBusiness Plan): BioBusiness

Brazil is a competition of business plans directed at the creation of companies in

biotechnology and Health. SUPERA supports the participants in the elaboration of

Business Plans and gives an award to the best. The 1st BioBusiness SUPERA involved

40 projects, the 2nd BioBusiness Brazil received support from nine technological

incubators and involved 50 projects (from 8 different States) and received an

award from ANPROTEC as the Best Project for the Promotion of Culture of

Innovative Enterprise of 2007. The two editions involved a total of 94 projects

registered for SUPERA and resulted in two incubated companies. The 3rd edition

will be launched in October of 2008 during the Biolatina and its goal will be the

prospection of 100 projects (Site www.concursobiotecnologia.com.br),

d) Program of support to the dissemination of Enterprising Culture on the

Ribeirão Preto Campus of USP. The objective of these actions is to encourage the

creation of new business by the transformation of the scientific knowledge

generated in the university into new products, with the simultaneous generation of

a working alternative for postgraduate students in the technological areas, and to

expand in a sustainable manner the number of new projects in the selection

process of SUPERA. These actions will have an effect on a medium-term basis and

aim at the possible future expansion of the incubator by the transfer of companies

to the Technological Park of Ribeirão Preto. The main actions are:

• To work with the postgraduate program of FMRP in order to offer the

discipline “Innovation and Intellectual Property” on as semestral basis developed in

partnership with FEA-RP, SUPERA and the Innovation Agency of USP;

• To replicate the discipline created by SUPERA in partnership with the Faculty

of Philosophy, Sciences and Letters of Ribeirão Preto, more specifically with the

Department of Chemistry, entitled “Technological Enterprise”. This discipline was

taught in the first semester of 2008 in the courses for the Baccalaureate in

Chemistry, Forensic Chemistry and Habilitation in Technological Chemistry,

Biotechnology and Agroindustry. The objective of the discipline is to present to the

student the enterprising and innovative environment of the Ribeirão Preto USP

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Campus, to encourage the transfer of the technologies produced at the university,

and to train the students in the basic tools needed to plan and set up in practice a

company based on technology in order to expand the number of new projects in

the selection processes of SUPERA.

e) National Program for the generation of new business (BRBiotec – Brazilian

Network of Biotechnology Companies). The general objective of BrBiotec is to act

as a relationship network and to operate as a physical and virtual business location

that will be located in strategic places in the country. It will promote the

integration among Brazilian biotechology companies, investors, education and

research institutions, investigators and the federal, state and municipal

government. This network has already received funds of R$ 400 thousand from the

Ministry of Health and is being executed by the incubators SUPERA, CIETEC and

BIORIO, which are articulating a partnership with NISP – NATIONAL INDUSTRIAL

SYMBIOSIS PROGRAM OF THE UNITED KINGDOM, in order to exchange experience

and the sustainable business development by transforming low value resources into

added value which result most of the time in innovative products.

f) First Enterprising Business Program (PRIME): SUPERA was one of the 18

incubators chosen by FINEP to act as a decentralized operator of this institution in

the PRIME Program. The program intends to invest R$ 12 million in incipient

businesses based on technology. The project of the company contemplated by the

Prime will be supported by two granting modalities operated by Finep. The total

financing amount will be of the order of R$ 240 thousand per company. These funds

will be liberated over a period of two years, with the first parcel of R$ 120

thousand coming from the Program of Economic Support of Innovation. In this

modality, the funds are not reimbursable. The second and last parcel will use

money of the Zero Interest Program, which involves the return of the loan in

twenty payments with no interest. The first R$ 120 thousand parcel will be

disbursed in the form of non-reimbursable economic subvention and therefore will

be tax-free. These funds can be used to hire technicians, administrators and

consultants. SUPERA will publish the edict for the selection of businesses interested

in receiving the subvention funds in November 2008.

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G) Detailed Description of the Proposing Group

The present proposal for constitution of the INCTC is a joint initiative of 112

professionals and students (researchers, postgraduate students, post-docs and

research technicians) organized in eleven research groups represented by their

respective coordinators. Figure 5 depicts this professional and academic universe.

The Project will be coordinated by Prof. Dr. Roberto Passeto Falcão, CNPq

researcher level 1A and full professor at University of São Paulo, who has the

following qualifications: graduation in School Medicine at University of São Paulo,

Ribeirão Preto (1966), doctorate in Medical Clinic at University of São Paulo,

Ribeirão Preto (1972), post-doctorate at Oxford University (1975 – 1976). Currently,

Dr. Falcão is associate professor of the Faculty of Medicine, University of São Paulo,

Ribeirão Preto. He has experience in Medicine area, with emphasis on Medical

Clinic and Hematology and Immunology, and he is involved mainly in the following

subjects: aplastic anemia, lymphocytes, myeloid and lymphoproliferative diseases.

Outstanding points of his CV: http://lattes.cnpq.br/0739840296573834

1. Vice-coordinator of Project CEPID-FAPESP Center for Research on Cell-Based

Therapy, it started in 2000, with a budget of US$ 3.830.000.

2. Head of Medical Clinic Departament of Medicine Faculty of Ribeirão Preto from

1997 to 2001. This department is the largest at FMRP, and includes 60

professors. Dr. Falcão is one of the leaders in scientific production of this unit

at USP.

3. President of Brazilian Hematology School from 1998 to 2005.

4. Coordinator of Hematology sector of Clinical Medicine Departament of FMRP,

and of Hematology Lab of Clinical Hospital of Ribeirão Preto since 1988. This

division has been responsible for the education of approximately 80 residents

and numerous professionals that currently develop professional or academic

activities all around Brazil or abroad.

5. Founder (1993) and Coordinator of Brazilian Hematology School, it is an

organization that offers continuing education courses in the field of

Hematology. Twenty-one courses spanning different areas of Hematology have

been offered. A partnership with the European School of Hematology was

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established in 2000, and since then it has allowed lecturers visitations and the

use of didactic material from that organization in tutorial courses.

6. Dr. Falcão has published 127 scientific articles listed in Lattes curriculum

system, 100 are listed in PubMed (falcão rp; passetto falcão r; passeto falcão r;

falcão r). He has been cited 666 times, and his h index is 14. His activities as

advisor have resulted in 5 Master's dissertations and 9 PhD theses. He has also

been the co-advisor in 3 Master's dissertations and 2 PhD theses. Currently, Dr.

Falcão supervises one post-doc, one PhD student, and two Master’s students. He

has presented 181 communications in meetings with published abstracts. He has

been granted 10 awards related to these works.

7. He has written 19 book chapters, and he is the editor, together with Marco

Antonio Zago and Ricardo Pasquini, of the book Hematologia e Fundamentos e

prática médica 1/1. ed. São Paulo: Atheneu, 2004. v. 1. 1081 which involves 50

collaborators.

8. Representative of SBPC at Conselho Nacional de Saúde

9. Member of the editorial board of the Periodical Brazilian Medic Association,

Einstein, Journal of Brazilian Hematology and Hemotherapy, and

Hematology/Oncology and Stem Cell Therapy

The following principal researchers also take part in this proposal: 2. Maria Angélica Miglino - researcher level 1A of CNPq. Maria Angélica Miglino has

received her PhD in Sciences (funcional anatomy: structure and ultra-structure)

from Universidade de São Paulo in 1985. Currently, Dr. Miglino is a full professor at

University of São Paulo. She has published 294 articles in specialized periodicals

and 366 works in meeting proceedings. She has published 5 book chapters and 2

books. She has 1 technique process and other 10 technical production items. Dr.

Miglino has participated in 23 meetings abroad and 59 in Brazil. Her activities as

advisor resulted in 23 Master's dissertations, 27 PhD theses. She has been a co-

advisor in 1 Master's dissertation, and in 2 PhD theses. In addition, she has advised

scientific initiation works in the Veterinary Medicine and Morphology fields. She has

been granted 8 awards and/or honors. She has coordinated 36 research projects

110

between 1993 and 2004. Currently, she is engaged in 24 research projects and

coordinates 22 of these. Dr. Miglino develops activities in the Veterinary Medicine,

with emphasis on animal reproduction. During her professional activities, she has

interacted with 941 collaborators in co-authorships of scientific works. The most

frequent terms used to contextualize her scientific, technological and artistic-

cultural in her Curriculum Lattes are: placenta, anatomy, morphology, bovines,

study, ruminants, dogs, arterial, plaque, and vascularization.

http://lattes.cnpq.br/0806064137922471

3. Lewis Joel Greene – researcher level 1A of CNPq. He is full professor at

Medicine Faculty of Ribeirão Preto, University of São Paulo, in the Department Of

Celluar Biology, Molecular and Pathogenic Bioagents. Dr. Greene is the Supervisor

of Center of Chemic Proteins at Blood Bank of Ribeirão Preto, where he develops

studies concerning the chemical, functional and structural characterization of

proteins, using traditional protein chemistry approaches and proteomic analysis.

The research lines developed by him comprise: a) Studies on the functional and

structural characterization of proteins during the proliferation and cellular

differentiation of hematopoietic progenitor cells and human tumor cells, using a

proteomic approach; and b) Chemical and biological characterization of biologically

active proteins using traditional protein chemistry methods. Dr. Greene has

published 112 articles in specialized periodicals, 243 works in meeting proceedings

including complete studies and abstracts, and 3 book chapters. His activities as

advisor have resulted in 20 Master’s dissertations and 21 PhD theses. In addition, he

has advised 2 scientific initiation works. http://lattes.cnpq.br/7810652486138159

4. Lygia V. Pereira, Ph.D., obtained her Bachelor of Physics from Pontifícia

Catholic University of Rio de Janeiro in 1988. She received her Master’s degree in

Biological Sciences (Biophysics) from Federal University of Rio de Janeiro in 1990,

and her Ph.D. in Biomedical Sciences from the Mount Sinai School of Medicine, City

University of New York in 1994. Currently, she is Associate Professor MS5 (livre

docente) at University of São Paulo, member of Science Academy of São Paulo

State, and she is a head of Molecular Genetic Lab of Bioscience Institute, USP. She

has experience in the field of Genetics, with emphasis in Human Molecular

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Genetics, and she is involved in the following subjects: animal models of genetic

disease, embryonic stem cells, epigenetic inheritance, and X chromosome

inactivation. She is author of two scientific divulgation books: “Seqüenciaram o

Genoma Humano... E Agora?” and “Clonagem: da Ovelha Dolly às Células-Tronco”,

Editora Moderna. Dr. Pereira has published 39 articles in specialized periodicals, 30

works in meeting proceedings including complete studies and abstracts, 7 book

chapters and 4 books. She has published 35 articles in magazines/newspapers. Her

activities as advisor resulted in 4 Master’s dissertations and 8 PhD theses. She has

received 9 awards and/or honors. http://lattes.cnpq.br/1550542923772116

5. Stevens Kastrup Rehen – CNPq researcher level 2. He is a Biologist (UFRJ,

1994), Master of Biological Sciences (UFRJ, 1996), and Ph.D. in Sciences (UFRJ,

2000), and he did a post-doc at University of California at San Diego, USA (2000 -

2003). He is an Associate Researcher at The Scripps Research Institute (California,

USA) since 2005, Fellow of Pew Foundation, and Adjunct Professor at UFRJ. Dr.

Rehen works with basic research. He investigates the mechanisms involved in the

neural differentiation of embryonic stem cells and induced pluripotent stem cells,

and also study the culture of these cells in large-scale. He has published 23

scientific articles in indexed scientific journals, 11 of these produced exclusively in

Brazil, in addition to 8 book chapters, 16 scientific divulgation articles, 11

comments on science in digital magazines, and 1 scientific divulgation book. Dr.

Rehen leads Laboratório de Neurogênese e Diferenciação Celular of Instituto de

Ciências Biomédicas at UFRJ. He has advised 8 scientific initiation students, and

two Master’s students. Currently, he supervises 1 researcher (post-doc), 6 PhD

students, 3 Master’s students and 5 scientific initiation students. In addition, Dr.

Rehen has acted as reviewer for 7 international periodicals, and is Affiliate Member

of Brazilian Science Academy. More details can be found at:


6. Wilson Araújo da Silva Jr. - CNPq researcher level 1D. He graduated in Biology,

Medical Modality (Biomedicine) at Pará Federal University 1989, and obtained his

Master's degree in Genetics from Medicine Faculty of Ribeirão Preto, University of

São Paulo in 1993. He received his Ph.D. in Genetics from Medicine Faculty of

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Ribeirão Preto, University of São Paulo, in 1999. Dr. da Silva did a post-doc in

Cancer Genetics at Ludwig Institute, Memorial Sloan-Kattering Cancer Center in

New York City, NY, USA. He is Associate Professor of Genetic Department of

Medicine Faculty, Ribeirão Preto – USP. He develops his research in the field of

Human and Medical Genetics, working mainly with the following subjects: DNA

polymorphisms, Bioinformatics, and Cancer Genetics. Dr. da Silva has published 79

articles in specialized periodicals and 80 works in meeting proceedings including

complete studies and abstracts. His activities as advisor have resulted in 9 Master’s

dissertations, 6 PhD theses, and he has advised 3 scientific initiation students. He

has received 5 awards and/or honors. http://lattes.cnpq.br/8655277884027052

7.Eduardo Magalhães Rego – CNPq researcher level 2. He graduated in Medicine at

Medicine Faculty of Ribeirão Preto, University of São Paulo, in 1988. He obtained

his doctoral degree in Medical Clinic from the same institution (1997), and he did a

post-doc at Memorial Sloan Kettering Cancer Center in New York, USA. Currently,

Dr. Rego is Associate Professor at University of São Paulo, and he works in the field

of Hematology. His research line focuses on acute leukemias, with special emphasis

on Acute Promyelocytic Leukemia. He has published studies that demonstrate that

the hybrid genes PML/RARa, PLZF/RARa, NPM/RARa and NuMA/RARa are able to

induce leukemia in transgenic mice. These transgenic models have been used for

the study of the leukemia molecular bases and for research on new therapeutic

approaches. Dr. Rego also studies the relationship between ribosome function and

cancer by analyzing mice that carry the mutations in the gene DKC1. His most

important works are: 1. Yoon A, Peng G, Brandenburger Y, Zollo O, Xu W, Rego E,

Ruggero D. Impaired control of IRES-mediated translation in X-linked dyskeratosis

congenita. Science. 2006 May 12;312(5775):902-6. 2. Rego EM, Ruggero D, Tribioli

C, Cattoretti G, Kogan S, Redner RL, Pandolfi PP. Leukemia with distinct

phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or

NPM/RAR alpha. Oncogene. 2006 Mar 23;25(13):1974-9. 3. Rego EM, Wang ZG,

Peruzzi D, He LZ, Cordon-Cardo C, Pandolfi PP. Role of promyelocytic leukemia

(PML) protein in tumor suppression.J Exp Med. 2001 Feb 19;193(4):521-29. 4. Rego

EM, He LZ, Warrell RP Jr, Wang ZG, Pandolfi PP. Retinoic acid (RA) and As2O3

treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the

distinct nature of the leukemogenic process induced by the PML-RARalpha and

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PLZF-RARalpha oncoproteins. Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10173-8

Dr. Rego published 50 articles in specialized periodicals, 162 works in meeting

proceedings including complete studies and abstracts, and 10 books. He has

participated in 91 meetings in Brazil and abroad. His activities as advisor resulted

in 5 Master's dissertations and 4 PhD theses. In addition, Dr. Rego has advised 4

scientific initiation works, and has received 10 awards and/or honors.


8. Júlio Cesar Voltarelli – CNPq researcher level 2. He graduated in Medicine at

Medicine Faculty of Ribeirão Preto, University of São Paulo (FMRP-USP) (1967 –

1972). He completed his residency in Medical Clinic and Hematology at Clinical

Hospital of FMRP-USP (1973-74), and obtained his Master’s (1975-1978) and

doctoral (1978-1981) degrees in Medical Clinic from FMRP-USP. Dr. Voltarelli

engaged in post-doctoral activities in the following institutions: University of

California at San Francisco, USA (1985-86), Fred Hutchinson Cancer Research

Center in Seattle, USA (1987-88), and Scripps Research Institute in San Diego, USA

(1999-2000). Currently, he is full Professor of Clinical Medicine of FMRP-USP, and

Coordinator of Divisão de Imunologia Clínica, Laboratório de Imunogenética (HLA),

and Unidade de Transplante de Medula Óssea of HC-FMRP-USP. He is a researcher

of Centro de Terapia Celular (CEPID-FAPESP), hosted at Centro Regional de

Hemoterapia of HC-FMRP-USP. Dr. Voltarelli has published 99 articles in specialized

periodicals, 41 works in meeting proceedings including complete studies and

abstracts, and 13 book chapters. His activities as advisor have resulted in 13

Master's dissertations and 4 PhD theses. He has received 6 awards and/or honors.

He has experience in the field of Clinical Immunology and Hematology, with

emphasis on: hematopoietic stem cell transplantation for autoimmune diseases and

malignant and benign hematological disorders; diagnosis and treatment of

secondary immunedeficiencies and rheumatic disorders; selection of donors for

renal and bone marrow transplantations. http://lattes.cnpq.br/2521841581315624

9. Flávio Vieira Meirelles – researcher level 2 of CNPq. He obtained his Doctor of

Veterinary Science degree from Universidade Estadual Paulista Júlio de Mesquita

Filho (1993), and his Master’s degree in Sciences Vétérinaires Option Reproduction

from Université de Montréal (1997). He received his Ph.D. in Genetics from

114

Universidade de São Paulo (1999). Currently, he is Associate Professor ms5 (livre

docente) at Universidade de São Paulo; ad hoc consultant for Fundação de Amparo

à Pesquisa do Estado de São Paulo, associate editor of Genetics and Molecular

Research, Sociedade Brasileira de Tecnologia de Embriões, and consultant for the

journals Heredity, Animal Genetics, and Genetics and Molecular Biology. Dr.

Meirelles has experience in the field of Genetics, with emphasis on Cytoplasmic

Inheritance, and is involved mainly with the following subjects: mtDNA, Bos

indicus, cattle, embryos and apoptosis. http://lattes.cnpq.br/6698246156573680

10. Dimas Tadeu Covas – CNPq researcher level 1D. He graduated in Medicine at

Universidade de São Paulo (USP) in 1982. He received his Master’s (1986) and

doctoral (1993) degrees from USP, and he obtained livre-docência from the same

University in 1999. Currently, Dr. Covas is Associate Professor of Faculdade de

Medicina de Ribeirão Preto/USP, President-Director of Fundação Hemocentro de

Ribeirão Preto, and Coordinator of Transference of Centro de Terapia Celular (CTC-

CEPID-FAPESP). He is a hematologist and hemotherapist, and he develops research

on the following subjects: molecular and cellular biology, stem cells, erythrocytic

and platelet antigens, viruses (HIV and HTLV), protein heterologous expression in

cellular systems in vitro. He is an active member of Sociedade Brasileira de

Hematologia e Hemoterapia and of Colégio Brasileiro de Hematologia, and he is

currently the treasurer of this organization. He is Associate Editor of Revista

Brasileira de Hematologia e Hemoterapia, and is one of the founders of Escola

Brasileira de Hematologia. In addition, Dr. Covas is member of Academia de

Ciências de Ribeirão Preto. He has also promoted the dissemination of science and

education, and organized postgraduate programs for teachers and elementary and

high school students. He has edited two books, and one of these was awarded

Prêmio Jabuti in 2007. Dr. Covas has published 75 articles in specialized

periodicals, 380 works in meeting proceedings including complete studies and

abstracts, 12 book chapters, and 10 books. He has participated in 121 meetings in

Brazil and abroad. His activities as advisor resulted in 18 Master's dissertations and

5 PhD theses. In addition, Dr. Covas has advised 2 scientific initiation works, and

has received 9 awards and/or honors. http://lattes.cnpq.br/5244926449437566

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11. Lawrence Charles Smith received his Doctor of Veterinary Medicine from

Universidade Estadual Paulista Júlio de Mesquita Filho at Jaboticabal in 1991. He

obtained his Master’s degree from Faculty of Sciences, Université d'Édimbourg in

1984, and a Ph.D. in Physiology and Genetics from Faculty of Sciences, Institute of

Animal Physiology and Genetics, in 1989. Currently, he is full Professor of Faculté

de médecine vétérinaire, Centre de recherche en reproduction animale. Dr. Smith

works mainly with the following subjects: Nucleus-cytoplasm interactions,

embryos, cloning. He has been involved in the organization of postgraduate courses

for teachers and elementary and high school students. He has edited two books,

and one of these was awarded Prêmio Jabuti in 2007. Dr. Smith has published 59

articles in specialized periodicals, and 13 book chapters. His activities as advisor

have resulted in 9 Master’s dissertations and 9 PhD theses.


12. João Palermo Neto holds a productivity scholarship level 1B from CNPq.

Currently, he is full Professor at Universidade de São Paulo, and consultant of

Fundação de Amparo à Pesquisa do Estado de São Paulo, Food And Agricultural

Organization of The United Nations, Fundação de Amparo à Pesquisa do Estado de

Minas Gerais, Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro,

Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul and Conselho

Nacional de Desenvolvimento Científico e Tecnológico. He has experience in the

field of Pharmacology, with emphasis on Neuropsychopharmacology, and his work

involves mainly the following subjects: CNS, diazepam, dopamine,

neuroimmunomodulation, innate immunity and stress. Dr. Palermo Neto has

published 137 articles in specialized periodicals, 304 works in meeting proceedings

including complete studies and abstracts, 21 book chapters and 2 books. He has

participated in 67 meetings in Brazil and abroad. His activities as advisor resulted

in 36 Master's dissertations and 25 PhD theses. In addition, he has advised 18

scientific initiation works, and has received 14 awards and/or honors.

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PhD students

Researches

Pos-doc (4)

Ms students (12)

Technicians (1)

PhD students

Researches

Pos-doc (1)

Ms students (2)

Technicians

PhD students

Researches

Pos-doc (1)

Ms students (5)

Technicians

PhD students

Researches

Pos-docs

Ms students

Technicians (1)

PhD students

Researches

Pos-docs (1)

Ms students

Technicians (1)

PhD students

Researches

Pos-doc

Ms students

Technicians

PhD students

Researches

Pos-doc

Ms students

Technicians

PhD students

Researches

Pos-doc

Ms students

Technicians

PhD students

Researches

Pos-doc (2)

Mestrandos (5)

Técnicos (2)

PhD students

Researches

Pos-doc (2)

Ms students (1)

Technicians (1)


Wilson Araújo da Silva Junior

Flávio Meirelles

Stevens Kastrup Rehen

Júlio César Voltarelli

Lygia da Veiga Pereira

Roberto Passetto F l ã

Dimas Tadeu Covas

Maria Angélica Miglino

Lewis Joel GreeneINCTC

(PI)

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H) Activities Specification that must be done by the Team members

Researchers (PI)

Subprojects

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30


Dimas Tadeu Covas


Lewis Joel Greene

Maria Angélica Miglino

Wilson Araújo da Silva Junior

Júlio César Voltarelli

Flávio Vieira Meirelles

Lawrence CharlesSmith

Lygia da Veiga Pereira

Stevens Kastrup Rehen

Klena Sarges Marruaz da Silva

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I) Mechanisms that will be used to promote the Interaction of

the Research Groups

The interaction between research groups will be happen in different

ways beginning in the researches projects since your conceptions,

multilaterally and complementarily (item H). The INCTC’s Coordination will

be composed of five important research centers and universities (FMVZ-SP-

USP, IB-USP, UFRJ, CTC, FZEA-USP). This collaboration will guaranty the

viability and development of project. Another interaction mechanism that

we will use it will be meetings each tree months and annual symposium with

national and international investigators.

The interactions day by day can be done using INCTC website

(www.hemocentro.fmrp.usp.br/projeto/inct/) and also meetings by phone,

Internet and/or videoconference can be done. We must emphasize that

there is already ongoing collaboration between the groups, independent of

the beginning of this project.

The groups will also interact in education programs and knowledge

transfer to society. It is expected a creation of post-graduate courses intra-

units (between USP units) and inter-units (between USP, UFRJ and the

National Center for Primates).

In the innovation project, the Enterprise incubator linked to the CTC

can provide full logistic support and knowledge for the interaction of

different groups with the productive sector (for example, studies about

economic viabilities, business plans, micro-company, and intellectual

property).

Special attention will be give to the group of the National Center for

Primate which needs intensive training in techniques of cultivation and

updated characterization cell. The interaction mechanisms in this particular

case include periods of Belém professionals training in other laboratories of

the group and also the displacement of investigators to Belém for training in

loco.

119

J) Forms of Interaction with Foreign Groups

The development of an institute of this port must have an

international collaboration. Our group has already collaboration with 5

international researches that are currently developing international

activities in collaboration with our team.

This institute aims, through face or non-face activities (video

conference), to keep a strong contact with abroad institutions.

The contact with the abroad Institution will be done meetings every

three months where the participants will be invite to participate by

videoconference. An annual meeting will be done we will organize a

Symposium where the international researches will make a short

presentation about their developed activities. In the other hand, the

national group will present the results by poster or oral presentation. This

annual symposium will be able to make the group close and to give an idea

of what have been doing in the different labs.

In some cases the visit of the international collaborator can be

extended to promote the development of short or medium term internships.

For each request of research visitation he will receive a member of

our group in your Lab to be training or to develop our project.

120

K) Definition of specific tasks for each entity

Institution Members involved

SubprojectsCoordinator

s

Responsible

Faculdade de Medicina de Ribeirão Preto/USP

3 25 Roberto Passetto Falcão

Hemocentro de Ribeirão Preto – Centro Regional de Hemoterapia HCFMRP/USP

31 3, 5, 10, 11,

14, 15, 22, 23 e 30

Dr Dimas Tadeu Covas

Faculdade de Zootecnia e Engenharia de Alimentos

5 8 e 9 Flavio Vieira Meirelles

Instituto de Biociências/USP 5 1, 6 e 7 Lygia da Veiga Pereira Faculdade de Medicina Veterinária e Zootecnia/USP

11 26 Maria Angélica Miglino


6 13 e 16 Wilson Araújo da Silva Jr

Instituto de Ciências Biomédicas da UFRJ

13 2 Stevens Kastrup Rehen


4 19 e 20 Eduardo Magalhães Rego


18 17, 21, 28 e

29 Júlio Cesar Voltarelli

Faculdade de Medicina Veterinária e Zootecnia/USP

11 26 João Palermo Neto

Faculdade de Medicina de Ribeirão Preto/USP 12 12, 18 e 24 Lewis Joel Greene

Centro Nacional de Primatas 8 4 e 27 Klena Sarges Marruaz da Silva

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L) Comparative Analysis between the Current and Expected

Statuses

The teams involved in this proposal have a large experience in the

subject chosen, as demonstrated by their publications, patent deposits, and

activities aimed at diffusing knowledge to the community. The ways with

which this proposal intends to improve the current status are: effectively

exchange previous experience; support Centro Nacional de Primatas

(National Primate Center) effectively, enabling it to became an important

center for preclinical studies involving nonhuman primates; structure two

stem cell banks (one for animal cells, and another for human cells);

establish a Preclinical Studies Center; develop methodologies to culture

embryonic and mesenchymal stem cells in large scale and under clinical-

grade conditions; develop methodologies and products that may generate

patents; perform two phase I and II clinical studies; diffuse the knowledge

obtained to the community; train human resources; and contribute to the

improvement of public education. Many of these activities are already

performed, but the differential is that their range and scale will be

increased. The most significant advancement, however, will be the

establishment of the Institute itself, which will connect the participant

teams and provide a powerful stimulus for the development of this area in

the country.

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M) Justified Budget

General

Datadescription TotalValue(R$) ConsumableMaterials 3,899,596.37

PermanentMaterials 3,547,600.40

TravelTickets 292,926.70

Scholarships 1,007,717.30

BibliographicMaterials 2,790.00

Travelsupport 152,469.95

8,903,100.72

Consumable Materials

Item Value(R$) Antibodies 96,000.00

OfficeMaterials 54,630.00

BloodCollectionMaterials 416,696.00

LaboratoryPlasticware 502,158.00

Reagents 63,000.00

ElectrophoresisReagents 450,000.00

CytogeneticsReagents 142,521.87

CellCultureReagents 1,039,360.50

Reagentsforexperimentswithanimalmodels 46,000.00

ProteomicsReagents 900,000.00

Solvents 176,260.00

LaboratoryGlassware 12,970.00

3,899,596.37

123

Permanent Materials

Item Quant Description

1 3 Refrigerated microcentrifuge

2 7 Refrigerated bench centrifuge

3 1 Refrigerated floor-standing centrifuge

4 11 Laminar-flow Hood

5 1 work station

6 1 Network server

7 2 Transfection equipments

8 2 Thermal cycler

9 1 Shaking incubator

10 4 Water bath

11 2 Freezer -20° C

12 1 Refrigerator 4° C

13 3 Horizontal electrophoresis system

14 2 Notebooks

15 2 PC

16 3 Printers

17 3 Vertical Electrophoresis system and accessories

18 1 Helical computerized tomography equipment

19 3 Magnetic stirrer for spinner flasks

20 33 Spinner flasks

21 2 Bioreactors

22 1 Automated cell counter

23 1 Filtration system

24 1 MILLI-Q BIOCEL System and accessories

25 02 Liquid nitrogen containers and accessories

26 01 digital pH meter

27 38 Automatic micropipettes and automatic repeating pipettes

28 2 Real time PCR machine

29 3 Nucleic acid quantification systems

30 2 Analytical balances

31 6 Mixers (stirrers, hotplate stirrers and vortexes)

32 1 GFP goggles

33 4 Inverted trinocular microscope

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34 1 Optical microscope with polarized light with image capture system

35 1 Digital camera for inverted microscope with USB connector

36 1 Inverted microscope Axio Observer D1 with accessories

37 2 Analogical and digital video camera

38 2 Trinocular microscope

39 1 Articulated arm base for stereomicroscope

40 1 Digital camera 1.4 MB with sensitivity to detect fluorescent light

41 1 Trinocular stereomicroscope

42 1 Simple binocular microbiological microscope

43 1 Light source for GFP (Green Fluorescent Protein) excitation

44 1 Fluorescence Stereomicroscope

45 1 C-mount 1X adapter for camera/microscope

46 1 Dapi/Hoescht/Amca/HiSky filter and cubes for all types of microscope

47 1 Software Multi-Species for use with BandView

48 1 Complete workstation for cytogenetics: HiSKY, G band and FISH

49 1 Light microscope

50 1 Image acquisition system for inverted microscope

51 1 Stereomicroscope

52 01 23 L autoclave system

53 01 Flow cytometer

54 07 Accessories for cell separation columns

55 09 CO2 Water-Jacketed Incubator

56 01 Shaker Incubator

57 01 Bacteriological incubator

58 02 Tri-gas incubator

59 02 -80°C Freezers

60 01 Microarray Scanner

61 01 Microarray Hybridization oven

62 01 Rotational chamber for hybridization oven

63 01 SureHyb Hybridization chamber

64 01 Femtojet microinjector

65 01 Femtojet Microinjetor Pedal

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66 01 Narashige Air Microinjector

67 01 Piezo Drill

68 01 Fyrite CO2/O2

69 01 Automatic gas exchange system

70 01 GFP excitation light source

71 01 DAKO hybridizer

72 01 Photographic documentation system for agarose gels

73 01 Microplate luminescence reader

74 01 Cryostat

75 01 Automated pipetting system for PCR reactions

76 05 Multimidia projector

77 05 Digital video camera

78 03 LCD TV

79 25 Display cases

80 5 Showcase

JUSTIFICATIONS

1) Refrigerated microcentrifuge

The refrigerated microcentrifuges are designed for processing of DNA and

RNA samples that will be utilized in the cloning and analysis of expression

of target genes. 02 equipments will be installed at Laboratório de

Hematologia of Hospital das Clínicas de Ribeirão Preto – USP under the

responsibility of Prof. Eduardo Magalhães Rego, and 01 equipment will be

installed at Laboratório de Biologia Molecular of Hemocentro de Ribeirão

Preto, under the responsibility of Dr. Simone Kashima Haddad.

Subprojects: 3, 7, 11, 15, 18, 19, 20, 25, 26, 27, 30.

2) 07 Refrigerated bench centrifugue

The refrigerated bench centrifuges are designed for processing samples of

leukemic cell lines and retroviral packing cells, and also for Ficoll density

126

separation of bone marrow stem cells to be used in the protocols related

to transplantation in the animal models involved in this project. 01 unit

will be installed at Laboratório de Hematologia of Hospital das Clínicas de

Ribeirão Preto – USP under the responsibility of Prof. Eduardo Magalhães

Rego; 02 units will be installed at Prof. Maria Angelica Miglino’s

laboratory (Faculdade de Medicina Veterinária-USP); 02 units will be

installed in Dr. Passeto’s laboratory (Hematologia, FMRP); 01 unit will be

allocated to Laboratório de Biologia Molecular of Hemocentro de Ribeirão

Preto and 01 unit to Dr Julio Voltarelli’s laboratory. Subprojects: 3, 4, 6,

7, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30.

3) 03 Refrigerated floor-standing centrifuge

This equipment is intended for processing bone marrow samples before

and after retroviral transduction that will be used in the transplantation

protocols involving monkeys. This equipment will be installed in the

Specific Pathogen Free Animal House under the responsibility of Prof.

Eduardo Magalhães Rego (Hematologia, FMRP). Subprojects: 3, 7, 11, 18,

19, 20, 25, 26, 27, 30.

4) 11 Laminar-flow Hood

The laminar-flow hood is designed for the protection of the product and

operator in the manipulations of biological materials and toxic substances

in the fields of medicine, pharmaceutical industry and microbiology (for

manipulation of non-pathogenic materials). This equipment is proper for

the manipulation of cell and biological materials in aseptic conditions. 01

unit will be installed at Laboratorio de Hematologia of Hospital das

Clínicas de Ribeirão Preto – USP and another one in the Specific Pathogen

Free Animal House, both under the responsibility of Prof. Eduardo

Magalhães Rego; 01 unit will be installed at Hemocentro de Ribeirão Preto

under the responsibility of Prof. Wilson Araújo da Silva Júnior; 01 unit will

be allocated to Centro de Quimica de Proteínas under the responsibiliy of

Prof. Lewis Joel Greene; 05 units to Faculdade de Medicina Veterinária-

USP under the responsibility of Prof. Maria Angélica Miglino; 01 unit to

127

Laboratório de Hematologia under the responsibility of Prof. Roberto

Passeto Falcão and one to Laboratório de Morfofisologia Molecular e

Desenvolvimento under the responsibility of Prof. Flávio Meirelles.

Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 26, 27, 28, 30.

5) 1 work station

This hardware avoids PCR contamination, and will be installed at Centro

de Química de Proteínas of Hemocentro de Ribeirão Preto under the

responsibility of Prof. Lewis Joel Greene. Subprojects: 8, 12, 17, 18, 21,

23, 24.

6) 1 Network server

The network server will be installed at Hemocentro de Ribeirão Preto for

better connectivity between laboratories. Subprojects: 2, 3, 4, 5, 6, 8,

10, 11, 12, 14, 15, 16, 17, 20, 21, 22, 23, 26, 27, 28, 29, 30.

7) 2 Transfection equipments

This equipment enables transfection of human cells with vectors and

double-stranded RNA. This equipment enables the transfection without

cell content destabilization, and allows the obtainment of higher cell

viabilities after transfection. 01 unit will be installed at Hemocentro de

Ribeirão Preto under the responsibility of Prof. Wilson Araújo da Silva

Júnior, and the other will be allocated to Laboratório de Morfofisologia

Molecular e Desenvolvimento under the responsibility of Prof. Flavio

Meirelles. Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19,

20, 22, 24, 26, 27.

8) 2 Thermal cycler

Used to DNA and cDNA amplification. 01 unit will be installed at

Hemocentro de Ribeirão Preto under the responsibility of Prof. Covas, and

the other will be allotted to Faculdade de Medicina Veterinária-USP under

128

the responsibility of Prof. Maria Angélica Miglino. Subprojects: 1, 4, 5, 6,

8, 9, 11, 19, 26, 27.

9) 1 Shaking incubator

The incubator will be used for growth of bacterial cultures. This

equipment will be installed at Hemocentro de Ribeirão Preto under the

responsibility of Prof. Dimas tadeu Covas. Subproject: 5.

10) 4 Water bath

This equipment will be used for thawing cryopreserved cells, warming up

reagents, and incubating samples with restriction and/or modification

enzymes. 02 units will be installed in the Specific Pathogen Free Animal

House under the responsibility of Prof. Eduardo Magalhães Rego; 01 unit

will be allotted to Faculdade de Zootecnia e Engenharia de Alimentos,

USP, under the responsibility of Prof. Flávio Meirelles, and one will be

allocated to Centro Nacional de Primatas Evandro Chagas under the

responsibility of Prof. Klena Sarges Marruaz da Silva Sarges Marruaz da

Silva. Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 11, 18, 19, 20, 25, 26, 27, 30.

11) 2 Freezer -20° C

For the cryopreservation of biological samples and reagents. Both

equipments will be installed at Laboratório de Hematologia, FMRP under

the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 3, 7,

11, 18, 19, 20, 25, 26, 27, 30.

12) 1 Refrigerator 4° C

Used to refrigerate biological samples and reagents. This equipment will

be allotted to Laboratório de Hematologia da FMRP-USP under the

responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 3, 7, 11, 18,

19, 20, 25, 26, 27, 30.

129

13) 3 Horizontal electrophoresis system

Equipment used to separate nucleic acids. 01 unit will be installed at

Laboratório de Biotecnologia of Hemocentro de Ribeirão Preto under the

responsibility of Prof. Dimas Tadeu Covas, and 02 units will be allocated

to Laboratório de Hematologia of FMRP-USP under the responsibility of

Prof. Eduardo Magalhães Rego. Subprojects: 3, 5, 7, 11, 18, 19, 20, 25,

26, 27, 30.

14) 2 Notebooks

In the itinerant actions and in courses conducted outside of museums, the

notebooks are fundamental to multimedia projections, performance of

Physics shows, and also to organization of databases and secretary

services. 01 notebook will be used by Prof. Jussara R. Ferreira at

Universidade de Brasilia, and the other will be used by Prof. Eduardo

Magalhaes Rego at Labotarório de Hematologia of FMRP-USP. Subprojects:

3, 7, 11, 18, 19, 20, 25, 26, 27, 30.

15) 2 PCs

These equipments are fundamental to diagramming, web design, image

treatment, and also to production of didactic material and databases that

use images. 01 unit will be used by Prof. Jussara R. Ferreira at

Universidade de Brasília, and 01 unit will be used by Prof. Eduardo

Magalhães Rego at Laboratório de Hematologia of FMRP-USP. Subprojects:

3, 7, 11, 18, 19, 20, 25, 26, 27, 30.

16) 3 Printers

Multifunctional printers are necessary for printing high-quality

photographs, and also to supply the graphic needs of the projects. 01

unit will be installed at Laboratório de Biotecnologia of Hemocentro de

Ribeirão Preto, under the responsibility of Prof. Dimas Tadeu Covas; 01

unit will be allotted to Centro Nacional de Primatas Evandro Chagas,

under the responsibility of Prof. Klena Sarges Marruaz da Silva, and 01

unit will be allocated to Laboratório de Hematologia of FMRP-USP under

130

the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 2, 3, 4,

5, 6, 7, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27,

28, 29, 30.

17) 3 Vertical Electrophoresis system and accessories

This equipment is necessary to separate protein extracts through

mass/charge before their transference to nitrocellulose or PVDF

membranes to do Western-blots. 02 units will be used by Prof. Stevens K.

Rehen at Instituto de Biociências of UFRJ, and the other will be used by

Prof. Eduardo Magalhães Rego at Laboratório de Hematologia of FMRP-

USP. Subprojects: 2, 3, 5, 6, 7, 11, 18, 19, 20, 23, 24, 25, 26, 27, 30.

18) 1 Helical computerized tomography equipment

This equipment would be important to support the diagnosis of diseases

under study in subprojects related to stem cell therapy since it provides

precise three-dimensional images, which allow for the examination of

overlapping areas. This equipment also detects minor tissue alterations,

allowing for the early diagnosis of several alterations such as hemorrhage,

tumors, injuries, lung parenchymal alterations, etc. The aforementioned

equipment would be, thus, an important tool for the detection of

problems and, by extension, for the success of animal treatments

involving cell therapy. This equipment will be allocated to Instituto

Nacional de Primatas Evandro Chagas under the responsibility of Prof.

Klena Sarges Marruaz da Silva. Subprojects: 4, 6, 20, 26, 27.

19) 03 Magnetic stirrer for spinner flasks

This equipment is used to stir cells cultured in suspension in spinner

flasks. The spinner flasks, as well as the stirrer, should be kept in a CO2

incubator. 2 units will be allocated to Instituto de Ciências Biomédicas of

Universidade Federal do Rio de Janeiro under the responsibility of Dr K.

Stevens K. Rehen, and 1 unit will be allotted to Hemocentro de Ribeirao

Preto under the responsibility of Dr T. Dimas T. Covas. Subprojects: 2,

5, 6, 23, 24.

131

20) 33 Spinner flasks

Spinner flasks are small-scale flasks with different volumes where the

stem cells can be cultured in suspension. The spinner flasks will be placed

inside a CO2 incubator and stirred by a magnetic stirrer also placed inside

the incubator. 24 flasks will be allocated to Instituto de Ciências

Biomédicas of Universidade Federal do Rio de Janeiro under the

responsibility of Dr Stevens K. Rehen, and 9 of them will be allotted to

Hemocentro of Ribeirao Preto under the responsibility of Dr T. Dimas

Covas. Subprojects: 2, 5, 6, 23, 24.

21) 02 Bioreactors

This equipment will be used for expanding stem cells in a higher scale and

with precise control of culture parameters such as pH, dissolved oxygen

concentration, and temperature. 1 unit will be allocated to Instituto de

Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the

responsibility of Dr Stevens K. Rehen, and 1 unit will be allotted to

Hemocentro de Ribeirão Preto under the responsibility of Dr Dimas T.

Covas. Subprojects: 2, 5, 6, 23, 24.

22) 01 Automated cell counter

For cell number determination of several samples in an automated and

reproducible way. This equipment will be installed at Instituto de

Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the

reponsibility of Prof. Stevens K. Rehen. Suproject: 2

23) 01 Filtration system

This system will be used to filter samples containing cell and

microcarriers for the determination of the respective dry weights. This

system will be allocated to Hemocentro de Ribeirão Preto under the

responsibility of Prof. Dimas T Covas. Subproject: 2, 23.

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24) 01 MILLI-Q BIOCEL System and accessories

This Ultrapure water production system releases high quality water with

minimum ionic and organic contaminants and pyrogens, which is

mandatory for culture, expansion and maintenance of stem cells. This

equipment will be allocated to Instituto Evandro Chagas under the

responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects 4, 6, 20,

26, 27.

25) 02 Liquid nitrogen containers and accessories

For cryopreservation of cell and biological samples at low temperatures

(around -100ºC to -196ºC). One unit will be located at Instituto Evandro

Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva, and

the other at Faculdade de Zootecnia e Engenharia de Alimentos under the

responsibility of Prof. Flávio Meirelles. Subprojects 1, 4, 5, 6, 8, 9, 11,

19, 20, 26, 27.

26) 01 digital pH meter

Physiological pH values range from 7.15 to 7.4 and, hence, keeping

culture medium pH close to these values is important to maximize cell

expansion in vitro. This equipment will be kept at Instituto Evandro

Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva.

Subprojects 4, 6,20, 26, 27.

27) 38 Automatic micropipettes and automatic repeating pipettes

Pipettes are necessary to transfer various volumes of liquids in virtually

all laboratory techniques (molecular biology, cell culture, flow

cytometry). These equipments will be used by all groups, and they were

requested by the following coordinators: Dr. Wilson Araújo da Silva

Junior, Dr. Julio Voltarelli, Dr. Dimas Tadeu Covas, Dr. Roberto Passetto

Falcão and Dr. Flávio Meirelles. Subprojects 1, 5, 7, 8, 9, 11, 12, 13, 14,

15, 16, 17, 19, 20, 21, 22, 23, 25, 26, 28, 29, 30

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28) 02 Real time PCR machine

Real time quantitative RT-PCR is an essential technique to quantify gene

expression. It is very useful whenever it is necessary to verify differences

in gene expression among samples and/ or situations. Any model which

needs to verify messenger or microRNA expression will use quantitative

RT-PCR. It is also needed to validate gene expression values obtained by

microarrays, and to confirm the presence or absence of molecular

markers on stem cells. These equipments will be allocated to Instituto de

Biociências of UFRJ – RJ and at Laboratório de Genética Molecular of

Hemocentro de Ribeirão Preto. Subprojects: 2, 6, 7, 13, 16, 17, 20 e 24).

29) 03 Nucleic acid quantification systems

The preparation of samples to be analyzed by RT-PCR consists in

preparing cell samples, extracting their RNA, quantifying and verifying

the quality of RNA, and synthesizing complementary DNA. Quantification

and verification of the quality of this RNA are absolutely necessary to

obtain correct results by quantitative RT-PCR. The equipments used to

quantify and verify the quality of RNA samples also are also useful to

perform these tasks on protein and DNA samples. The latest models of

these equipments use very small sample amounts, which is very important

to study rare cell populations such as stem cells. These machines can be

used by all groups working with cells, DNA, RNA or proteins. These

equipments will be allocated to Instituto de Biociências of UFRJ – RJ,

Laboratório de Biotecnologia, and to Laboratório de Genética Molecular

of Hemocentro de Ribeirão Preto. Subprojects: 2, 5, 6, 13, 16, 17, 20, 23,

24.

30) 02 Analytical balances

Analytical balances are essential to correctly prepare laboratory

solutions, which should contain the precise amount of each reagent. This

type of balance can be used by all groups, and was requested by Dr.

Klena Sarges Marruaz da Silva from Instituto Evandro Chagas and Dr. Julio

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Voltarelli from Faculdade de Medicina de Ribeirão Preto. Subprojects 4,

6, 7, 17, 20, 21, 23, 26, 27, 28, 29).

31) 06 Mixers (stirrers, hotplate stirrers and vortexes)

Adequate mixing of reagents, solutions and samples is required in order

to obtain correct results. These equipments will be allocated to

Laboratório de Biologia Molecular and Centro de Químicas de Proteínas of

Hemocentro de Ribeirão Preto, and to Laboratório de Hematologia of

FMRP-USP. Subprojects: 5, 7, 8, 11, 12, 14, 15, 17, 18, 19, 20, 21, 22, 23,

24, 28, 29, 30, 12, 14, 15, 17, 20, 21, 22, 23, 28, 29 e 30.

32) 1 GFP goggles

These goggles bear specific green filters for macroscopic observation of

GFP animals. It will be allocated to Faculdade de Zootecnia e Engenharia

de Alimentos under the responsibility of Prof. Flávio Meirelles.

Subprojects: 1, 8, 9, 20, 27.

33) IInverted trinocular microscope

For routine observation of cell cultures as well as the analysis of cell

differentiation. They will be allocated to Departamento de Hematologia

of FMRP-USP (1 unit) and Faculdade de Medicina Veterinária-USP (3

units). Subprojects: 3, 4, 6, 7, 8, 9, 10, 11, 13, 18, 19, 20, 22, 25, 26,

27, 30.

34) 1 Optical microscope with polarized light and image capture

system

For acquisition and analysis of high quality digital images. This equipment

will be allocated to Faculdade de Medicina Veterinária-USP under the

responsibility of Profa. Maria Angélica Miglino. Subprojects: 21, 23, 26,

29.

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35) 1 Digital camera for inverted microscope with USB device

For capture of images obtained on inverted microscope. It will be

allocated to Faculdade de Medicina Veterinária-USP under the

responsibility of Prof. Maria Angélica Miglino. Subprojects: 21, 23, 26, 29.

36) IInverted microscope Axio Observer D1 plus accessories

For observation, analysis and documentation of each step of cell

development. It also allows for the exhibition of the images on the

screen. It will be allocated to Instituto Evandro Chagas under the

responsibility of Dr. Klena Sarges Marruaz da Silva. Subprojetos: 4 e 27.

37) 2 Analogical and digital video camera

Image capture device for trinocular microscope. It will be used in the

educational projects under the responsibility of Prof. Jussara R. Ferreira

of Universidade de Brasília.

38) 2 Trinocular microscope

Equipment necessary for acquisition of the images that will be used to

build image databases; to prepare didactic materials for lectures,

courses, and expositions; to prepare divulgation material (flyers, folders,

games, CDs, etc) for the popularization of knowledge. It will be used in

the educational projects under the responsibility of Prof. Jussara R.

Ferreira from Universidade de Brasília.

39) 1 Articulated arm base for stereomicroscope

This equipment is required for a trinocular stereoscope. It will be used in


Ferreira of Universidade de Brasília.

40) 1 Digital camera 1.4 MB with sensitivity to detect fluorescent light

This equipment is required for acquisition of the images used to build the

image database; preparation of didactic material for lectures, classes,

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and expositions; preparation of diffusion material (pamphlets, folders,

handouts, games, CDs, etc.) for diffusion of knowledge. It will be used in


Ferreira from Universidade de Brasília.

41) 1 Trinocular stereomicroscope

This equipment is necessary to visualize biological material (plants,

animal’s cells), rocks, sand and other materials during lectures on the

museum center in Ribeirão Preto, or even during itinerant actions. It will

be used in the educational projects under the responsibility of Prof.

Jussara R. Ferreira from Universidade de Brasília.

42) 1 Simple binocular microbiological microscope

For cell counting using the Neubauer chamber during cell culture

procedures. It will be installed at Hemocentro de Ribeirão Preto under

the responsibility of Prof. Dimas Tadeu Covas. Subprojects: 5, 8, 10, 11,

12, 14, 15, 20, 21, 22, 23.

43) 1 Light source for GFP (Green Fluorescent Protein) excitation

This equipment will be used for gross analyses of expression of the GFP

reporter gene by live organisms. This equipment will equip Laboratório de

Morfofisiologia Molecular e Desenvolvimento, of Faculdade de Zootecnia e

Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles,

and will be used frequently in subprojects 1, 8, 9, 20, and 27

44) Fluorescence Stereomicroscope

This equipment is necessary to visualize the GFP reporter protein in

cultured cells or live organisms. This equipment will equip Laboratório de

Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e

Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles.

Subprojects: 1, 8, 9, 20, 27.

45) C-mount 1X adapter for camera/microscope

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It will be used to couple the CCD camera to the fluorescence microscope,

for use with the softwares SKYView or SpectraView. It will be installed at

Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto

under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19,

20, 25.

46) Dapi/Hoescht/Amca/HiSky filter and cubes for all types of

microscope

These filters are necessary for the observation of fluorescent substances

used in cytogenetic preparations. This equipment will be installed at



20, 25.

47) Software Multi-Species for use with BandView

This software will be useful for detailed analyses of cytogenetic findings

that reflect genetic instability at the chromosome level in materials from

humans and rodents, and also to build an image bank for future analyses

and publication of the results. This equipment will be installed at



20, 25.

48) Complete workstation for cytogenetics: HiSKY, G-banding and FISH

This equipment will be used to investigate numeric and structural

chromosomal abnormalities in metaphase cells of patients with chronic

lymphoid leukemia (CLL), and will contribute to related projects that

include cytogenetic analyses. It will be installed at Laboratório de

Hematologia of Faculdade de Medicina de Ribeirão Preto under the

responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, 25.

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49) Light microscope

This equipment will be used to investigate numeric and structural

chromosomal abnormalities in metaphase cells of patients with CLL, and

will contribute to related projects that include cytogenetic analyses. It

will be installed at Laboratório de Hematologia of Faculdade de Medicina

de Ribeirão Preto under the responsibility of Prof. Roberto Passeto

Falcão. Subprojects 19, 20, 25.

50) Image acquisition system for inverted microscope

This equipment will be used for observation and documentation of cell

culture conditions during the experiments. It will be installed at

Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas

Tadeu Covas. Subprojects: 5, 8, 10, 11, 12, 14, 15, 20, 21, 22, 23.

51) Stereomicroscope

This equipment is necessary for the selection of labeled cloned cells "in

situ". It will be installed at Hemocentro de Ribeirão Preto under the

responsibility of Prof. Dimas Tadeu Covas. Subprojects: 5, 8, 10, 11, 12,

14, 15, 20, 21, 22, 23.

52) 23 L autoclave system

This equipment is necessary for sterilization and cleaning of materials

used in cell culture. This equipment will be allocated to Faculdade de

Medicina Veterinária-USP under the responsibility of Prof. Maria Angélica

Miglino. Subprojects: 21, 23, 26, 29.

53) Flow cytometer

Under any circumstances, such as after cell culture or sorting, adult or

embryonic stem cells must be identified with the highest precision

possible. Immunophenotypical identification using 4-color flow cytometry

provides, in addition to precision, the possibility of simultaneous

identification of 4 independent cell markers. This equipment will be

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allotted to Instituto Nacional de Primatas Evandro Chagas under the

responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects: 4, 6, 20,

26, 27.

54) Accessories for cell separation columns

These accessories are required for assembly of the immunomagnetic cell

separation system, enabling handling of four cell types at the same time.

Cell isolation by MACS has been extensively used to obtain pure cell

populations, and it yields cells that maintain their physiological status

with high cell viability. The cells are immunomagnetically labeled and

then passed through columns held in a magnetic field. This equipment

will be allocated to Instituto Nacional de Primatas Evandro Chagas under

the responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects: 4, 6,

20, 26, 27.

55) 09 CO2 water-jacketed incubator

The CO2 incubators will be used for cell culture. Considering the

projected volume of work necessary for the generation of the expected

results, the acquisition of CO2 incubators for stem cell culture will be

indispensable. The incubators will be installed in laboratories of the

following units/institutions: Ciências Biomédicas/UFRJ-RJ, Faculdade de

Medicina de Ribeirão Preto/USP, Hemocentro de Ribeirão Preto, and

Faculdade de Medicina Veterinaria e Zootecnia/USP. Subprojects: 2, 6, 7,

11, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30.

56) 01 Shaker Incubator

This shaker incubator will be used for expansion of bacteria. It will be

allocated to Laboratório de Biotecnologia of Hemocentro de Ribeirão

Preto. Subproject: 5.

57) 01 Bacteriological incubator

This equipment will be used to culture and select bacterial clones in solid

medium, allowing for the obtainment of plasmids used for functional

140

analyses of microRNAs. It will be installed at Laboratório de Biologia

Molecular of Hemocentro de Ribeirão Preto. Subproject: 15.

58) 02 Tri-gas incubators

These equipments will be used for cell culture in different types of cells

in hypoxia conditions, to verify the cells’ behavior under these

conditions. One of them will be installed at Faculdade de Zootecnia e

Engenharia de Alimentos of Universidade de São Paulo – Pirassununga, and

the other will be allotted to Laboratório de Terapia Celular of

Hemocentro of Ribeirão Preto. Subprojects: 1, 8, 9, 10, 19, 26.

59) 02 -80°C Freezers

The -80°C freezer enables storage and maintenance of reagents and cell

samples. They will be installed at Laboratório de Hematologia of

Faculdade de Medicina de Ribeirão Preto and at Faculdade de Zootecnia e

Engenharia de Alimentos of Universidade de São Paulo – Pirassununga.

Subprojects: 1, 7, 8, 9, 18, 19, 20, 26.

60) 01 Microarray Scanner *

The acquisition of a scanner for DNA microarray (CGH array) analyses is

necessary to read microarray slides, and for the evaluation and

identification of genes. This equipment will be allocated at Laboratório

de Hematologia of Faculdade de Medicina de Ribeirão Preto. Subprojects:

3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, 29.

61) 01 Microarray Hybridization oven *

This equipment is necessary to incubate microarray slides during the

hybridization step. It will be allocated to Laboratório de Hematologia of

Faculdade de Medicina de Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15,

16, 17, 19, 20, 21, 25, 29.

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62) 01 Rotational chamber for hybridization oven *

This equipment is necessary for the microarray hybridization oven. It will

be allocated to Laboratório de Hematologia of Faculdade de Medicina de

Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, 29.

63) 01 SureHyb Hybridization chamber *

This equipment is necessary for the microarray hybridization oven. It will

be allocated to Laboratório de Hematologia of Faculdade de Medicina de

Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, 29.

* These equipments will provide the infrastructure for establishment of

the microarray methodology that will serve all subprojects that involve

large-scale gene expression analyses.

64) 01 Femtojet microinjector

This device is necessary to perform microinjection/micromanipulation of

ova or embryos of different species. It will equip Laboratório de



Subprojects: 1, 8, 9, 19, 26.

65) 01 Femtojet Microinjetor Pedal

This device is necessary to perform microinjection/micromanipulation of

ova or embryos of different species. It will equip Laboratório de



Subprojects: 1, 8, 9, 19, 26.

66) 01 Narashige Air Microinjector

This equipment will be used for aspiration of the cytoplasmatic content of

ova. It will be allocated to Laboratório de Morfofisiologia Molecular e

Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos

142

under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19,

26.

67) 01 Piezo Drill

This device will be used in techniques involving microinjection and

micromanipulation of embryos reconstructed in the project. It will be

allocated at Laboratório de Morfofisiologia Molecular e Desenvolvimento

of Faculdade de Zootecnia e Engenharia de Alimentos under the

responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, 26.

68) 01 Fyrite CO2/O2

This equipment is necessary for the maintenance of adequate gas

concentrations inside the incubator, and it is essential for the adequate

production of in vitro embryos. It will be allocated at Laboratório de



Subprojects: 1, 8, 9, 19, 26.

69) 01 Automatic gas exchange system

This equipment is necessary for the uninterrupted supply of gas for

incubators since it allows the use of two gas cylinders. It will be allocated

to Laboratório de Morfofisiologia Molecular e Desenvolvimento of

Faculdade de Zootecnia e Engenharia de Alimentos under the

responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, 26.

70) 01 GFP Model exciting power pack: FHS/LS-1B

This equipment will be used for evaluation of macroscopic expression of

reporter genes in living organisms. It will be allocated at Laboratório



Subprojects: 1, 8, 9, 19, 26.

143

71) 01 DAKO hybridizer

This system is necessary for hybridization of slides for molecular

cytogenetics (FISH and SKY). It will be allocated to Laboratório de

Hemtaologia of FMRP-USP under responsibility of Prof. Roberto Passeto

Falcão. Subprojects 19, 20, 25.

72) 01 Photographic documentation system for agarose gels

This system will be used for documentation of the initial tests for

optimization of the conditions of the amplification reactions. In this case,

the equipment requested will have 100% national technology, with

excellent construction quality and easy maintenance. It will be allocated

to Instituto de Biociências of UFRJ under responsibility of Prof. Stevens K.

Rehen. Subprojects 2, 6, 23.

73) 01 Microplate luminescence reader

This equipment will be used for the MycoAlert (Lonza) luminescent assay.

Furthermore, a luminescence reader can be used for a large number of

expression assays, in which the activation capacity of the promoter of a

specific gene can be measured along the time, or upon exposure to some

factor. It will be allocated to Instituto de Biociências of UFRJ under

responsibility of Prof. Stevens K. Rehen. Subprojects 2, 6, 23.

74) 01 Cryostat

A cryostat is necessary for the confection of sections of frozen tissue. It

will be allocated to Instituto de Biociências of UFRJ under responsibility

of Prof. Stevens K. Rehen. Subprojects 2, 6, 23.

75) 01 Automated pipetting system for PCR reactions

This system will be used for large scale pipetting in classic and real-time

PCR reactions. This equipment will also be used for nucleic acid

extraction. It will be allocated to Laboratório de Biologia Molecular of

Hemocentro de Ribeirão Preto, under responsability of Prof. Dimas Tadeu

144

Covas. Subprojects: 3, 5, 7, 8, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22,

23, 25, 26, 28, 29, 30.

76) 5 Multimedia projectors

These projectors are necessary for projections during itinerant museum

activities, as well as in extramural courses. It will be used in the

educational projects under the responsibility of Prof. Jussara R. Ferreira

from Universidade de Brasília.

77) 5 Digital video camera

These video cameras are necessary for documentation of lectures,

courses, and itinerant activities. It will be used in the educational

projects under the responsibility of Prof. Jussara R. Ferreira from

Universidade de Brasília.

78) 03 LCD TVs

These are necessary for projections during itinerant museum activities, as

well as in extramural courses. It will be used in the educational projects

under the responsibility of Prof. Jussara R. Ferreira from Universidade de

Brasília.

79) 25 Display cases

This demountable furniture will be used as showcases during the activities

of the participant museums. It will be used in the educational projects

under the responsibility of Prof. Jussara R. Ferreira from Universidade de

Brasília.

80) Showcases - 5

This equipament will be used for traveling exhibition around the country.

145

N) Potential for the Generation of Patents

Trials and Patents deposited/obtained by the proponent group

Process Title Year of the deposit of the process

PI

PI9603267-7 Quimioluminescence diagnosis test for infections by T cruzi 2007

Dimas T. Covas Luiz R Travassos Igor C Almeida

018080023071 Method of animal cloning from apoptotic cells and its use 2008 Flávio Meirelles

PI0775561-7 Process of stem cells obtention from orbicular lip cells, composition and use.

2007

Irina Kerkis Maria Rita Passos Bueno Daniela F Bueno Mayana Zatz

PI0501037-3

Process of isolation, purification and determination of crotamine aminoacid sequences and its use as vectors for condution of genetic material

2005

Irina Kerkis Alexandre Kerkis E. B. Oliveira G-Radis Baptista M. A Hayashi Lygia da Veiga Pereira T. Yamana

PI0502668-7

Process for obtaining stem cells, cell concentration, process for stem cell differentiation of cells, differentiated cells, different cells concentration, use of stem cells for treatment or prevention of diseases and stem cells bank

2005

Irina Kerkis Alexandre Kerkis M. A Hayashi H. F Cerruti

Total 5

The INCTC project has a big potential to generate patents and

products. Specifically in the areas of isolation, culture, establishment of

new lineages, productive process based on GMP criteria (good

manufacturing you practice), development of drugs use, tissue engineering

and regenerative therapy both in animals as in humans. As showed in figure

4, the generation of products on gene therapy area should pass for distinct

phases and the potential of generate patents is not the same in all of these

phases. The phases with bigger potential of patentability are phases II and

III. In Brazil, in general, the groups of research that are dedicated to the

study of stem cells and cell therapy are still found in phase I. In this project,

we are proposing activities that are distributed for phases I to IV and

therefore we will be working in the streak of bigger patents generation

146

probability in process and products. Several participants of the project have

experience in the development of patents and in the production of

technological process that need intellectual protection. Dr. Dimas Covas, by

example has 3 patients (one recorded and two in process of record), Dra.

Lygia Pereira has one patent recorded, Dra. Irina Kerkis has another three

recorded patents, Dr. Flávio Meirelles has several technological process

developed, the same happens with Dra. Maria Angelical Miglino. Therefore,

the group possess experience in the generation of technological products

that will be able to originate patents.

The question of the patents record of the products and process

generated will be directed with the aid of the USP Innovation Agency,

specialized in these questions, and also of the Foundation Hemocentro of

Ribeirão Preto, directly, or by its Incubator of Companies. Another point

that needs attention is the intellectual protection concerns of the copyright

of the publications and educational stuff produced in the printed or

electronic form (copyright) and to the protection of marks. The Hemocentro

has a sector of his Legal Consultancy specially focused in these aspects and

zealous of the record of the material produced.

147

O) List of the Projects Financed over the Last 5 years

COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE(R$) VALUE (US$)

PROF. DR. ROBERTO PASSETTO FALCÃO CNPQ

MONOCLONAL B CELLS LYMPHOCYTOSIS IN FIRST-DEGREE RELATIVES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

2007 - 2009

45,000.00

PROF. DR. DIMAS TADEU COVAS FINEP

CLONING AND EXPRESSION OF HUMAN BLOOD CLOTTING FACTORS VIII AND IX IN MAMMALIAN CELLS

21/11/2005 21/11/2008 01.05.0691.00 2556/05 370,000.00


CONSOLIDATION OF THE CELL CULTURE LABORATORY FOR MOLECULAR AND PROTEOMIC STUDIES OF DENDRITIC AND STEM CELLS

26/08/2005 26/08/2009 01.05.0466.00 0777/05 522,082.00


SCALING OF THE PRODUCTION OF RECOMBINANT FACTORS VII AND IX IN BIOREACTORS AND PRECLINICAL ASSAYS IN HEMOPHILIC MICE

19/12/2007 19/12/2009 01.07.0652.00 0927/07 2,384,565.82


BIOLOGICAL AND MOLECULAR ANALYSIS OF PLURIPOTENT SOMATIC STEM CELLS IN TISSUE REPAIR

04/12/2007 04/12/2009 01.07.0581.00 0156/07 434,050.00


INFRASTRUCTURE FOR THE GENERATION AND MANIPULATION OF GENETICALLY MODIFIED ANIMAL MODELS IN THE STUDY OF HUMAN DISEASES

25/09/2006 25/09/2008 01.06.0598.00 1081/06 742,800.00


DEVELOPMENT OF ANIMAL MODELS FOR THE UNDERSTANDING OF THE ROLE OF MESENCHYMAL STEM CELLS AND PERICYTES IN TISSUE REPAIR AND TUMOR VASCULARIZATION

AWAITING AGREEMENT 343,000.00

148

COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) VALUE (US$)

PROF. DR. DIMAS TADEU COVAS CNPQ

ISOLATION, CHARACTERIZATION, CULTURE, EXPANSION AND EVALUATION OF THE IN VIVO AND IN VITRO VASCULOGENIC POTENTIAL OF ADULT PLURIPOTENT STEM CELLS WITH CAPACITY FOR ENDOTHELIAL DIFFERENTIATION

01/03/2008 A 01/03/2009

558137/2008-3 54,158,80


EVALUATION OF THE GENE EXPRESSION OF ENDOTHELIAL PROGENITOR CELLS ISOLATED FROM UMBILICAL CORD BLOOD

26/12/2007 TO 25/12/2009

480770/2007-7 56,000.00


EVALUATION OF GENE EXPRESSION IN ENDOTHELIAL PROGENITOR CELLS ISOLATED FROM UMBILICAL CORD BLOOD AND BONE MARROW

17/07/2008 TO 17/07/2010

501541/2008-0 11,592.24

PROF. DR. DIMAS TADEU COVAS BNDES UMBILICAL CORD BLOOD BANK AWAITING

LIBERATION 872,220.00

PROF. DR. EDUARDO MAGALHÃES REGO FAPESP

DETERMINATION OF THE EFFECT OF INACTIVATION OF THE GENE CAATα ENHANCER’S LIGAND PROTEIN IN THE HEMATOPOIESIS OF IML-RARA TRANSGENIC ANIMALS

01/11/2001 TO 31/10/2003

2001/08693-8 182,890.33

PROF. DR. EDUARDO MAGALHÃES REGO CNPQ

IN VITRO AND IN VIVO ANALYSIS OF THE ANTILEUKEMIC ACTIVITY OF TOCOPHEROLS IN ACUTE PROMYELOCYTIC LEUKEMIA

01/03/2005 TO 01/03/2007

481911/2004-1 47,800.00

PROF. DR. EDUARDO MAGALHÃES REGO CNPQ

ANALYSIS OF THE ROLE OF THE TGFβ PATHWAY AND OF THE THERAPEUTIC POTENTIAL OF ITS INHIBITOR HALOFUGINONE IN ACUTE PROMYELOCYTIC LEUKEMIA

01/02/2008 TO 31/01/2010

484873/2007-5 80,000.00

PROF. DR. MARCO ANTONIO ZAGO FAPESP CELL THERAPY CENTER 2000 TO

2008 1998/14247-6 9,022,029.65 3,830,769.53

149


PROF. DR. MARCO ANTONIO ZAGO FAPESP TECHNICAL RESERVE FOR INFRASTRUCTURE 01/08/2007

31/07/2009 2007/54816-0 129,955.01 PROF. DR. MARCO ANTONIO ZAGO FAPESP TECHNICAL RESERVE FOR CONNECTION TO

THE ANSP NETWORK 01/08/2007 31/08/2008 2007/55689-2 26,982.00

PROF. DR. LEWIS JOEL GREENE FINEP

STRUCTURAL AND FUNCTIONAL PROTEOMICS APPLIED TO THE BIOMEDICAL AREA

14/12/2007 14/12/2009 01.07.0595.00 0875/2007 720,000.00

PROF. DR. LEWIS JOEL GREENE

FINEP AND FAPESP

SUPPORT TO THE PROTEOMIC NETWORK OF THE STATE OF SÃO PAULO

October 2007 to October/2009

140,000.00

PROF. DR. JULIO CÉSAR VOLTARELLI FINEP

HEMATOPOIETIC STEM CELL TRANSPLANTATION IN AUTOIMMUNE DISEASES

2004-2008

400,000.00

PROF. DR. JULIO CÉSAR VOLTARELLI CNPQ

HEMATOPOIETIC STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES AND NEURODEGENERATIVE DISEASES

01/10/2005 01/10/2008 552266/2005-1 395,949.55

PROF. DR. MARIA ANGÉLICA MIGLINO FAPESP

STUDY OF THE PLASTICITY OF IMMATURE MULTIPOTENT STEM CELLS DERIVED FROM HUMAN DENTAL PULP: AN ANIMAL MODEL. A BRIGE FOR CELL THERAPY

22/03/2007 to 21/03/2009

130,000.00

PROF. DR. MARIA ANGÉLICA MIGLINO CNPQ RESEARCH SUPPORT, UNIVERSAL CNPQ

PROJECT 2006 – 2008 35,000.00

PROF. DR. MARIA ANGÉLICA MIGLINO FAPESP

EXPRESSION OF GREEN FLUORESCENT PROTEIN (GFP) AS A MARKER OF CELLS OF FETAL ORIGIN IN GESTATIONS OF BOVINE CLONES

2006 – present date

05/52676-1

250,000.00

PROF. DR. LYGIA DA VEIGA PEREIRA FAPESP

ANALYSIS OF THE PATTERN OF CHROMOSOME X INACTIVATION IN HUMAN EXTRA-EMBRYONIC TISSUES

8/2005 – 2/2008

50,000.00

150


PROF. DR. LYGIA DA VEIGA PEREIRA FAPESP

CLINICAL VARIABILITY IN AN ANIMAL MODEL OF MARFAN SYNDROME – IDENTIFICATION OF PHENOTYPE-MODIFYING GENES.

11/2006 – 12/2009

142,000.00 38,000.00

PROF. DR. LYGIA DA VEIGA PEREIRA CNPQ ESTABLISHMENT OF NEW HUMAN

EMBRYONIC STEM CELL LINES 1/2006 – 8/2008 250,000.00

PROF.. DR. LYGIA DA VEIGA PEREIRA FAPESP

CREATION OF AN ANIMAL MODEL OF MARFAN SYNDROME BY HOMOLOGOUS RECOMBINATION IN EMBRYONIC STEM CELLS

10/2000 – 12/2004

200,000.00 60,000.00

PROF. DR. STEVENS KASTRUP REHEN CNPQ

CHARACTERIZATION OF THE IMPORTANCE OF LYSOPHOSPHATIDIC ACID FOR THE CULTURE OF NEURAL STEM CELLS DERIVED FROM THE CEREBRAL CORTEX OF MICE AND HUMANS

2005/2007

26,615.00


CHROMOSOMAL INSTABILITY DURING DIFFERENTIATION OF EMBRYONIC STEM CELLS TO MOTOR NEURONS

2008/2010

22,000.00


CONTROL OF ANEUPLOIDY AND CELL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

2005/2007

200,000.00

PROF. DR. STEVENS KASTRUP REHEN FAPERJ

PROPAGATION OF EMBRYONIC STEM CELLS IN BIOREACTORS IN ORDER TO INCREASE THE SCALE FOR TRANSPLANTS IN NEURODEGENERATIVE DISEASES

2007/2008

79,000.00


CONTROL OF CHROMOSOME INSTABILITY AND NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

2007/2008

16,000.00

151



CHARACTERIZATION OF THE THERAPEUTIC POTENTIAL OF STEM CELLS IN A PRECLINICAL MODEL OF PARKINSON’S DISEASE: MECHANISMS OF NEUROGENESIS AND FUNCTIONAL COMPARISON OF EMBRYONIC AND ADULT CELLS

2008/2010

400,000.00

PROF. DR. WILSON ARAÚJO DA SILVA JUNIOR FAPESP

IDENTIFICATION O AND FUNCTIONAL CHARACTERIZATION OF MOLECULAR MARKERS INVOLVED IN THE PROCESS OF FORMATION OF HEAD AND NECK TUMORS BY ANALYSIS OF THE DIFFERENTIAL PATTERN OF METHYLATION

464,712.00

59.616.00

PROF. DR. WILSON ARAÚJO DA SILVA JUNIOR FAPESP SEARCH FOR MARKERS OF AGGRESSIVENESS

IN HEAD AND NECK TUMORS

451,842.48

126.572,85

DR.SIMONE KASHIMA HADDAD CNPQ

QUANTITATIVE PROFILE OF MICRORNAs IN POPULATIONS OF T LYMPHOCYTES OF HTLV-I-INFECTED PATIENTS

01/10/2006 30/09/2008 475091/2006-0

38,600.00

PROF. DR. JOSÉ CÉSAR ROSA FAPESP

STUDY OF THE MICROSOME AND CYTOSOL FRACTIONS OF B LYMPHOCYTES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA – A DIFFERENTIATED PROTEOMIC ANALYSIS

2005-2007

10,648.00


STUDIES OF POST-TRANSLATION MODIFICATIONS IN PROTEINS DIFFERENTIALLY EXPRESSED IN THE CELL LINES MELAN-A, TM1 AND TM5. A MURINE MODEL OF MELANOMA PROGRESSION.

2006-2008

1,040.000.00


BIOLOGICAL EFFECTS AND PHARMACEUTICAL APPLICATIONS OF LECTINS

2008-2012

1,114,859.19

152


DR. KLENA SARGES MARRUAZ DA SILVA

UNESCO/CENP-IEC/SVS-MS

IDENTIFICATION AND CONTROL OF ZOONOTIC ENDOPARASITOSES IN NON-HUMAN PRIMATES MAINTAINED AT THE NATIONAL PRIMATE CENTER – SVS/MS

February 2005 to April 2006

50,400.00


OPAS/CENP-IEC/SVS-MS

CLINICAL AND EPIDEMIOLOGICAL MONITORING OF NON-HUMAN PRIMATES SEROPOSITIVE FOR VIRAL HEPATITIS MAINTAINED AT THE NATIONAL PRIMATE CENTER – SVS/MS

July 2006 to 02 October 2006

15,632.00


UNESCO/IEC/SVS-MS

IMPLANTATION OF A NEW METHOD FOR LABORATORY ANIMAL REARING AND RECOLONIZATION OF THE RODENT AND LAGOMORPH STOCK OF THE ANIMAL HOUSE OF THE INSTITUTO EVANDRO CHAGAS – IEC- SVS/MS

From February 2008 to the present date

47,.380.00

21,960,116.07 4,125,606.38

153

P) Formal Agreement by the Institutions Involved

176

Prof. Dr. Dimas Tadeu Covas Associate Professor FMRP/USP DD. President Director Fundação Hemocentro de Ribeirão Preto

September 15, 2008

Dear Prof Dimas,

I am delighted to accept your invitation to be a collaborator on the Project from CNPq Edital No 15/2008 ‐ entitled “National Institute of Science and Technology”, and thus continue our productive collaboration that began two years ago when one of your researchers visited my lab to learn the LM‐PCR technique. I am pleased to hear that a publication utilizing data generated with this technique is being written at the present time.

Important to the current project, we have a great deal of experience with Mesenchymal and Hematopoietic Stem Cells, as well as gene modification of stem cells using murine retrovirus and lentivirus vector systems. Moreover, we have developed two large animal models which we are using to study the in vivo distribution and differentiative capacity of specific human stem cells: 1) a line of Hemophilia A sheep; and 2) the human/sheep xenograft model.

Based on our prior collaborative productivity and our experience with in vivo models of stem cell transplantation/biology, I would be glad to collaborate with you and add my expertise to several of your sub‐projects.

Sincerely,

Graça Duarte de Almeida-Porada,MD,PhD Professor Director of Graduate Studies Department of Animal Biotechnology University of Nevada, Reno Mail Stop 202 Reno NV 89557-0104 Phone:(775)-784-6755 (775)-784-8048 LAB Fax: (775)-784-1375 [email protected]

University of Oxford

Joanna Poulton Professor and Honorary Consultant in Mitochondrial Genetics Tel: 01865 221067 Fax: 01865 769141 E-mail: [email protected]

Nuffield Department of Obstetrics & Gynaecology, University of Oxford, John Radcliffe Hospital, Women’s Centre, Oxford, OX3 9DU, U.K.

29/09/2007 Dr Flavio Vieira Meirelles Professor Livre Docente Faculdade de Zootecnia e Engenharia de Alimentos Universidade de Sao Paulo Dear Professor Meirelles, Thank you very much for your invitation to be a collaborator at the Project from CNPq Edital No 15/2008 – entitled National Institute of Science and Technology, which I am delighted to accept. We have studied segregation of pathogenic mtDNA mutants in tissue culture and in the human germline, carrying out the first oocyte-based studies in humans. These are essential for genetic management of these families. Hence the results from current proposal will be of great interest to us. We will train your excellent student, Marcos Roberto Chiaratti, in the methods of human mtDNA and nucleoid quantitation using fluorescence microscopy that we have developed. I look forward to hearing that the application has been funded. Yours sincerely

Joanna Poulton Professor and Honorary Consultant in Mitochondrial Genetics 179

180

Q) Institutional Contribution

Institution Human Resources

Laboratorial infrastructure

Total (R$)

Instituto de Biociências/USP 198,000.00 2,500,000.00 2,698,000.00

FMVZ 100,000.00 1,146,950.00 1,246,950.00 FEZEA-Pirassununga-USP 472,440.00 2,050,000.00 2,522,440.00


LABORATORY INFRASTRUCTURE (EQUIPMENT) TOTAL (R$)

Cell Biology 261,770.08 Gene Transfer 523,491.83 Flow Cytometry 653,634.13 Freezing of Hematopoietic Precursor Cells 1,834,650.99 Molecular Biology 855,449.37 Molecular Genetics 1,530,988.76 H.L.A. Laboratory 322,696.57 Protein Chemistry 319,360.12 Total 6,302,314.85

HUMAN RESOURCES MONTHLY

TOTAL (R$)

NO. OF MONTHS

TOTAL VALUE (R$)

Salaries and charges 44,541,47 60 2,672,488.20

CONSTRUCTION FUNDHERP TOTAL (R$)

Laboratory of Experimental Studies on Animals 1,470,000.00 Laboratory of Biotechnology 4,800,000.00 Total 6,270,000.00

181

INFRASTRUCTURE

Centro Regional de Hemoterapia – Blood Center of Ribeirão Preto

Hospital das Clínicas - Faculdade de Medicina de Ribeirão Preto/USP

Classrooms:

• Green classroom - (72 m²): unit with controlled temperature, it has

modulated tables with individual chairs, 80 people capacity,

equipped internet and multimedia system.

• Yellow classroom – (22 m²): unit with controlled temperature, it has

individual school chairs, 30 people capacity, equipped with mobile

multimedia system.

Amphitheaters:

• Red – (146 m²): with controlled temperature, it has individual chairs

with mobile arms, 180 people capacity, equipped with multimedia

system, internet and sound system.

• Blue – (62 m²): with controlled temperature, it has individual chairs

with mobile arms, 50 people capacity, equipped with multimedia

system, internet and sound system.

Informatic Center:

• (47 m²) Informatic central unit, it has: 1) Central Server, with an

optic disc unit to record and read, 16 DIMM memory module , HP

U320 SCSI 73 GB two Hard drives, 12 disc units of 72,8 GB, one unit of

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magnetic tape Ultrium 448, HPXV v1 – Model HP RP 4440 operational

system; 2) PA-RISC serie 9000 server, with 2 memory modules of 512

MB, with unit of magnetic tape Leasg 16-00, HP-UX V.11 – Model HP

L2000 operational system; 3) PA-RISC serie 9000 server, with disc

unit of 2 GB, two external dics of 2 GB – HP K210/128 model; 4) Data

bank: a) Oracle 10G Database Standard, platform HP-UXPA-RISC; b)

“Progress” system; 5) 2 Servers with 1024 MB memory, disc unit of 72

GB, HP DL 380 model; 6) Net system Hub – Model 3 COM Super Stack;

7) No-Break, nominal power de 10 KVA automatic; personal

computer, scanners and printers, among others.

Laboratories:

• Cryobiology I laboratory – (71 m²): this unit has support,

hematopoietic progenitor cell freezing and storage areas (umbilical

cord blood). It has the following equipments: sterile connection

device, microcomputer, pipettes, preservation chamber with embryo

freezer accessory to blood bags storage, storage system cryoplus,

dielectric manual sealer, hemoseal automatic sealer for blood bags,

laminar flow, automatic cell counter, -20C freezer, -86C freezer, 25

kg high pressure cylinder, water purification system to produce type 1

water, CO2 incubator, phmeter, optic inverted microscopy, thermal

hydrometer, cryogenic container for transportation, conservation

chamber, analytical balance, plasma extractor, frezenius universal

sealer, storage system “bioarchive”, low speed refrigerator

centrifuge, among others.

• Cryobiology II laboratory – (41 m²): laboratorial area. It has basically

the following equipments: inverted microscopy, colored monitors,

micro pipettes, electrophoresis chambers, vacuum pampers,

183

automatic electrophoresis system, refrigerator, laminar flows,

centrifuges, incubators, printers, colored table scanner, angle rotor,

freezer, video digital, duplex refrigerator, magnetic stirrer,

automatic thermo cycler, phmeter, incubator, advanced filtration

system, microcomputers and printers, among others.

• Quality Control Laboratory: (44 m²): laboratorial area. It has

basically the following equipments: coagulometer, precision

pipettes, refrigerators, microscopes, phmeters, water baths, freezer,

centrifuges, spectrophotometer, laminar flow, among others.

• H.L.A. Laboratory – (22 m²): laboratorial area. It has basically the

following equipments: thermo cycler, flow cytometer, platform,

microscope, water-bath, centrifuge, pipettes, digital camera,

electrophoresis chamber, balance, tubes stirrer, freezer

refrigerators, laminar flow, incubator, electrophoresis system,

microcomputer and printer.

• Cell Culture Laboratory – (68 m²): laboratorial and room culture

area. It has basically the following equipments: binocular optic

microscope, CO2 container, precision pipettes, freezer (-86°), phase

contrast inverted microscope, vortex, CO2 incubator, water bath,

refrigerated centrifuge, semi automatic photo-micrography system,

N2 container, laminar flow, binocular microscope, phmeter,

refrigerators, inverted microscope, analytical balance, water

distiller, electrophoresis system, bar code reader, laminar flow,

among others.

• Molecular Biology Laboratory: (94 m²): laboratorial area. It has the

following basic equipments: electrophoresis power supply, water

distiller, water bath, video copy processor, micro plates

184

homogenizator, pipettes, vacuum pump, microscopic image

acquisition system, transilluminator, laminar flow, incubator,

centrifuge, duplex refrigerator, automatic thermo cycler, sample

concentrator, freezer, spectrophotometer, speed vac with high

capacity components, microcomputers and printers, among others.

• Cellular Biology Laboratory: (67 m²): laboratorial area. It has the

following basic equipments: refrigerators, electrophoresis system,

printers, pipettes, chambers, spectrophotometer, cellmax pump

station, scintillations counter and micro plates, freezer, centrifuges,

microscope, water bath, laminar flow, microcomputers, printers,

among others.

• Flow Cytometry Laboratory: (47 m²): laboratorial area. It has the

following basic equipments: flow cytometry, pipettes, refrigerators,

vacuum pump, facstation power macintosh station, facsorting

interface, micro pipettes, centrifuges, microcomputers, printers,

among others.

Molecular Genetics Laboratory: (47 m²): unit composed by laboratorial

area. This laboratory have the following basic equipments: projector,

multimedia, automatic thermocyclers, freezers, VDS imaging system,

vertical laminar flow, pipettes, refrigerators, , hit bath, micro

centrifuges, magnetic stirrers, plate sealers, incubator for bacterial

culture, multi-screen mini protean, micro plates systems, sonicator,

dry thermo blocks, vertical and horizontal electrophoresis apparatus,

automatic sequencers, scanner HP Scanjet, micropipettes, digital

thermo mixer, personal computers, notebooks, desk jet, among

others.

Bio-informatics Laboratory: (25 m²): unit composed by informatics area.

This laboratory has the following basic equipments: personal

185

computers, desk scanners, desk jets, multimedia projector, among

others.

Search and Study Laboratory: (34 m²): unit composed by area of

informatics search. This laboratory has the following basic

equipments: personal computers, desk jets, etc.

Library: (42 m²): this unit have 1,160 books, 4,000 periodic and journals,

19 references sets (printed and digitalized), thesis, dissertations and

monographs, guides, 09 electronic signatures, 03 journals signatures,

annals and collection of scientific production of docent group, etc.

Audio-visual Sources: (33 m²): unit composed by area for elaboration

and production of bibliographic material and multimedia images. This

area has the following basic equipments: personal computers, desk

scanners, desk jets, plotter, camera camcorder, CD and DVD recorder

machine, portable projectors, etc.

• Nursing services – (76 m²) – this unit is compose by areas for nursing

management, administrative support, aphaeresis, transfusion room,

medical clinics, screening of donors and blood collection. This area

has the basic equipments: computers, printers, sphignomanometer,

aphaeresis systems, sealer blood bag, chairs for aphaeresis, infusion

bombs, urgency cars, vacuum clean surgical, etc.

• Social service – (21 m²) – this unit has areas for social support to

donors and ambulatory patients. It has the basic equipments:

computers, printers, scanner, video camera, photo camera, television

and DVD player.

• Laboratory of Hematology – (622 m2): this unit is composed of

administration area, laboratory and cell culture room. It has the basic

equipments: refrigerator, freezers –20 and – 80 C, centrifuges with

and without refrigeration, speed-vac centrifuge, cytocentrifuge,

186

microwave, hybridization oven, hybridization oven for microarray,

analytical balance, pHmeter, geneQuant, spectrophotometer, ice

machine, machine for cell freeze, bench refrigerate incubator,

thermo cycle, Real Time PCR system, dry bath, water bath, water

purification system, laminar-flow hood, rack negative isolator, CO2

incubator, horizontal shaker, tubes shaker, magnetic stirrer with and

without heating, power supply for electrophoresis chamber, vertical

and horizontal electrophoresis chambers, tissue drill, scanner, photo

documentation system, autoclave, cell counter/analyzed, pipettes,

computers and printers.

• Biocenter – (1.006 m²) – this unit is composed of areas for HLA and

biotechnology laboratories and hereditary anemias, Cell therapy,

Cytogenetic and Immunofluorescency, Science Education House and

support rooms.

• Laboratory of Biotechnology and hereditary anemias – It has the

basic equipments: microwave, shakers, water bath, incubator and

analytical balance.

• Laboratory of Cell therapy – It has the basic equipments:

microscopy, pippets, refrigerated centrifuge, freezer, water bath,

Laminar-flow Hood class 2, cell counter, CO2 incubator, Nitrogen

liquid container, electrophoresis chambers, phmetrer, analytical

balance, electrophoresis power and freezer -86C.

• Laboratory of Cytogenetic and Immunofluorescence – This

laboratory has the following basic equipments: microscopy,

fluorescent source, incubator, safety cabin, vortex, cell counters.

187

• Science house and Museum and Laboratory of Science Education –

this unit is composed of areas for education activities. It has the basic

equipments: computers, printers, plotter, scanner, DVD player,

cassette player, video cameras, photo cameras, multimedia system,

televisions, microscopy, electronic keyboard, notebook and

refrigerator.

• Laboratory of Animal Studies – (722,45 m²) – this unit has 2 floors for

animal manipulation, studies and chirurgic procedures. This unit is

under construction.

• Biotechnology Laboratory – (5.438 m²) – this unit has 6 floors for

highly specialized laboratories equipped for cell culture,

cryopreservation, serology, NAT, FVIII production. Project finalized

and approved, awaiting the release of resources to start the

construction.

• Animal Manipulation Room – (54 m²) – This unit is composed of areas

for animal manipulation. It has the basic equipments: safety cabin,

freezer, centrifuge and shelves for animal cases.

• Animal house – Faculdade de Farmácia: This unit is SPF (specific

pathogen-free), and is composed of 3 rooms for mice and rats. It has

the basic equipments: iron shelves and a “pass-through”, autoclaves,

ventilated breeding racks, shelves for maintenance of approximately

300 couples of matrixes of mice in expansion, a total of 8 lines,

providing the scientific community an average of 500 mice per month

and a total of 40 couples (matrixes) of rats Wister.

188

• Center for Chemical proteins – (460 m²) – this unit has areas for

sample preparation, electrophoresis, administration office and

meeting rooms for support. It has the basic equipments: tubes stirrer,

magnetic stirrer, electrophoresis power, refrigerator, hypercassete,

phmeter, and electrophoresis evaluation system, production system

of ultrapure water and pre-treatment of water.

Universidade de São Paulo

Instituto de Biociênicas/ Departamento de Genética e Biologia Evolutiva

• Cell culture room: is equipped with the following basic

equipments: laminar flow,CO2 incubators, Refrigerate Centrifuge,

Inverted microscope, Lupe, warm water baths, Micro-centrifuge,

Freezer –70C, Nitrogen liquid containers, electrophoreses

apparatus with agarose and polyacrilamide gel, 2 PCR machines

(24 and 96 wells), balances, incubators, pHmeters, shaking,

electronic pippettes, microwave, computers with internet access,

scanner and printers.

• Laboratory Animal Facility: This sector is located at the

Immunology Department and has already been working for 8 years.

This structure gives support to maintainance of mouses and rats in

pathogen-free conditions. The laboratory possess central air

conditions with 100% air renew, area with wash machines for

cages and water dispensers, ultrasound system for nozzle cleaner,

2 high volume autoclave and wash and dry machine for clothes,

uniforms for the employees and users All the material (cages, lids,

drinking trough and food) is autoclaved. Rooms of animal keeping

have 21 +- 2 °C temperature, with 12 light and 12 of dark. The

laboratory animal facilities are useful for routine genetic and

sanitary controls (virology, parasitology and bacteriology).

189

Universidade Federal do Rio de Janeiro

Instito de Ciências Biomédicas

• Laboratory of Neurogenesis and Cell Differentiation (LANDIC),

ICB UFRJ: has the follow basic equipments: Dissection and surgery

lupe, inverted microscope, regular microscopes, laminar flows,

CO2 incubators, orbital shakers, Millipore water production,

nitrogen container, refrigerator, freezers –20°C, freezer –70°C,

thermostatic baths, dissections instruments, refrigerated

centrifuges, (tubes of 15/50mL), microcentrifuges Eppendorf

5402; thermocyclers, electrophoresis powerpack and horizontal

and vertical chamber, electric Teflon plaque with termostate for

in situ hybridization, balances, incubators, pHmeters, shakers,

pipettes, microwave and others like Macintosh microcomputer, PC

computers, scanner and printers with internet access, automatic

cariotyping system BandView, stereotaxic device for mouse and

rats, fluorescent light microscopy with phase contrast, Axiocam

digital camera with software installed in a PC, microtome

equipment, cryostat and paraffin microtome.

190

Instituto Evandro Chagas Centro Nacional de Primatas

• This area is located on Ananindeua campus in Belém and is

comprised of management administration, storage room, library,

employee office, geoprocessing laboratory, scientific and

academic development room, scholarship program and quality

management. There is a Committeee of biosecurity, ethics and

research in human and also another one related to animals,

technical and scientific consul, and finally quality management.

Basic equipments: surgery, autoclaves, embebbed material,

microtome, stain material and equipment, light microscope,

Freezer -70°C, thermocycler, ELISA, serum biochemistry and 4D

ultrasound facilities.


Laboratory of Molecular Morphophysiology (LMMD) at Departmento de

Ciências Básicas da Faculdade de Zootecnia e Engenharia de Alimentos

• As infrastructure, the LMMD laboratory presents a built area of 280 m2

equipped with cell culture room with incubators, microscopes and

also molecular biology equipments, including Real time PCR, laser

image reader for different application as “differential display” PCR

and macro-array. The LMMD also has another area denominated

Animal Neonatology Unit (200 m2) that is currently under

construction (FINEP budget) and is designated to the large scale

production of transgenic mammals.


Faculdade de Medicina Veterinária e Zootecnia

• Laboratory of Microscopic Anatomy and Immunohistochemistry

191

This laboratory is equipped with distiller, oven for drying materials,

dispenser for paraffin, sputter coating machine, critical point, microtomes,

water bath, refrigerators for samples storage, microscopes, ultrasonic

cleaner, computers and a fume hood. It offers technological conditions to

support research in the area of organic light-microscopy, preparation of

tissues, molds, models and several staining techniques. It develops

microvascular techniques, preparation of molds and vascular models,

confection and analyses. It is provided with an imaging system for

microscopy with 10 trinocular microscopes, one interconnected with dual

observation system (Leica® industries Mod DME system) and acquires images

for television, using the trinocular tube for Leica microscopy, with adapter

C-Mount cameras for the video Color 1 / 3 "CCD Sansung® SDC-313 with

higher resolution of 480 lines-TV. It offers services to the general public

such as: preparation and staining of tissue for histological and

immunohistochemistry research; whole mount preparations, confection of

molds and vascular models for electron microscopy; preparation of stains

and fixative solutions for tissues; confection and preparation of semi-thin

and mesoscopic sections. The Laboratory was used to support the course of

practical expertise in Biology of Development and Embryonic stem cells in

2006.

• Laboratory of cell culture

Equipped with CO2 incubator, Leica® microscope adapted in laminar flow

(MINIFLOW) to analyze embryos of different species with guarantee of no

contamination of biological material, laminar flow (Veco®), inverted

microscope connected to an image system, refrigerator, water bath,

centrifuge and -80°C freezer, providing conditions to developing research

involving cell culture, and counts with materials such as bottles, falcon

tubes (15 and 50ml), tips, disposable pipettes, plates of various sizes,

filters, culture medias and supplements.

192

• Laboratory of Electron Microscopy

Equipped with a Scanning Electron Microscope (Leo - 435 VPZeiss), and a

Transmission Electron Microscope (MET) model Magni 268D (FEI Company -

Philips), equipped with a system for image analysis SIS DOCU MET, digital

camera 268, working kilovoltage between 40 and 100 KV, which increases

vary from 25 to 280.000X. It is also equipped with water cooling unit,

especially developed for biological sciences, in addition to two Ultra

microtomes (Leica), sputter coating machine (Balzers), a unit of Critical

Point and other equipments needed for sample preparing. It is adequate for

the analysis of different types of tissues and samples. The scanning electron

microscope has the ability to work with samples dehydrated or not, metallic

or not, ensuring speed, efficiency and low cost of processing. Performs the

preparation and analysis of electron micrographs; development and

enhancements of electron micrographs; Preparation and ultra cuts for

transmission electron microscopy; preparation and analysis of the material

for scanning electron microscopy; preparation of material for transmission

electron microscopy

• Surgery Center

It offers to the researchers the possibility of pre-clinical tests on different

types of animals. Equipped with surgical table, cupboard for medicines,

surgical focus, oxygen cylinder, trichotomy machine, and anesthetic

equipment HB Hospital (Galanti).

193

R) Time Table

Subproject Names 1º year 2º year 3º ano 4º ano 5º ano 1. Assessment of the epigenetic

stability of embryonic stem cell lines established under different conditions

2. Propagation of human embryonic stem cells in bioreactors in order to increase the scale for transplants in degenerative diseases

3. Molecular basis of hematopoietic differentiation from embryonic stem cells

4. Culture of adult and embryonic stem cells of humans and animals for use in cell therapies

5. Gene modification of stem cells 6. Establishment of induced

pluripotent stem cell lines (iPS) in models involving large anuimals

7. Establishment of iPS 8. Autologous pluripotent cells

generated from differentiated somatic cells

9. Animal model for the study of intra- and interspecies mitochondrial inheritance

194

Subproject Names 1º year 2º year 3º ano 4º ano 5º ano 10. Investigation of the identity of

mesenchymal stem cells (MSC): comparison of cultures MSCs and pericytes

11. Assessment of the angiogenic potential and elucidation of the molecular interactions of MSC and endothelial cells in in vivo and in vitro co-culture systems

12. Comparison of protein expression in human MSC obtained from bone marrow and from the umbilical cord vein

13. Differences in in the expression of genes and of immunophenotypic markers in amniotic epithelial cells obtained in the presence and absence of components of animal origin

14. Isolation and functional characterization of pluripotent stem cells “side population”

15. Functional evaluation of microRNAs in the differentiation of human pluripotent mesenchymal stromal cells

195

Subproject Names 1º year 2º year 3º ano 4º ano 5º ano 16. Analysis of gene expression

during in vitro osteogenic differentiation in samples from patients with osteogenesis imperfecta

17. Gene, protein and functional analysis of MSC from patients with autoimmune diseases

18. Determination of the levels of some proteins related to the apoptotic process ans to the control of the cell cycle of normal hematopoietic precursor cells and leukemic precursor cells

19. Role of diskerin in the differentiation of hematopoietic cells

20. Model of acute leukemia in Corocebus aethiops (Cercopithecus aethiops) using hematopoietic stem cells transduced with a retroviral vector containing the hybrid gene CALM-AF10

21. Assessment of the therapeutic potential of MSC for the regeneration of skin ulcers caused by extensive thermal burns in an animal model

196

Subproject Names 1º year 2º year 3º ano 4º ano 5º ano 22. Use of genetically modified MSC

for the expression of factor IX in murine models of hemophilia B

23. Development of a bioprocess for the expansion of MSC in microcarriers

24. Functional proteomics: studies of the role of nucleofosmin in gliomagenesis

25. Cytogenomics tools applied to the investigation of chromosome instability in chronic lymphocytic leukemia

26. Center of Preclinical Studies and animal stem cell bank

27. Availability of neotropical primates (new world) and of the species Chlorocebus aethiops (old world species) for research with adult and embryonic stem cells as a biological model of cell therapy

28. Treatment of type 1 diabetes mellitus with infusion of MSC

29. Treatment of multiple sclerosis with hematopoietic stem cells: evaluation of the clinical response and of the immunologic mechanisms of action

30. Clinical assay: use of MSC for the treatment and prevention of graft-versus host disease in patients

197

S) Indication of Management Committée

The management committée will be composed of the main

coordinator of the Project (Prof. Dr. Roberto Passetto Falcão) and by one

member of each of the four groups associated to this Project:

• Faculdade de Medicina Veterinária de São Paulo – Profa. Dra. Maria

Angelica Miglino

• Faculdade de Zootecnia de Pirassununga – Prof. Dr. Flávio Vieira

Meirelles

• Instituto de Biociências da USP e UFRJ – Profa. Dra. Lygia Veiga

Pereira

• Faculdade de Medicina de Ribeirão Preto – Prof. Dr. Dimas Tadeu

Covas

198

T) Organization and Functional Structure of the Institute

Project Coordinatortor

External Advisory Committée

Administration and Support Manager

Purchasing

Administrative Support

Quality Control and Audit

Research Coordinator

Research Program

Research Laboratories

Education Coordenator

Middle‐high School

Technical Personnel

University Students Doctoral

Post‐ Doctoral

Training of Industry Partners

Technology Transfer

Public Sector

CRH

Private Sector

Small Business Incubator

Comittee Gestor

USP SP 1 FMVZ Preclinical trial “Core Facilites”

UFRGS

UFG UFTO

UFERSA

UNB

UNESP - DRACENA

UFPI

UDESC

USP – SP 2 IB

USP - PIRASSUNUNGA

UFRJ

USP–Ribeirão Preto

Belém

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national institute of science and technology in stem cell

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