Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

BASIC PRINCIPLES

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 1

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

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Aim of the courseTo provide theoretical background knowledge to complex tissue cultures

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Course contentsWeek Lecture Title

1 1 Basic principles

2 Stem cells (1)

2 3 Stem cells (2)

4 Stem cells (3)

3 5 Bioreactors (1)

6 Bioreactors (2)

4 7 Biomaterials (1)

8 Biomaterials (2)

5 9 Scaffold fabrication

10 Biocompatibility

6 11 Cell-Scaffold interaction

12 Biofactors

7 13 Controlled release

14 Biosensors

Week Lecture Title

8 15 Aggregation cultures

16 Tissue printing

9 17 Tissue repair (1)

18 Tissue repair (2)

10 19 Tissue repair (3)

20 Tissue repair (4)

11 21 Commercial products (1)

22 Commercial products (2)

12 23 Clinical trials (1)

24 Clinical trials (2)

13 25 Clinical trials (3)

26Research applications and drug testing

14 27 Ethical Issues

28 Economic significance

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What is tissue engineering?• Tissue engineering was previously known as a

subfield of engineering and biomaterials, but having grown in scale and significance tissue engineering has become a discipline of its own.

• Tissue engineering aims to regenerate or recreate specific tissues or full organs with maintained or recreated biological function (bone, cartilage, blood vessels, bladder, skin, etc).

• To achieve the aims tissue engineering uses a combination of techniques including cell culture, engineering bio-materials, biochemical and biophysical methods.

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Basic principles of tissue engineering

Monolayer cell cultureExpanded cell culture

Generation of a graft

Cells from a biopsy or resection

Grafting Biopsy

Culture in 3D conditions

(scaffold based or scaffold free)

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2D tissue culturesAdherent Non-Adherent

Suspension

Pelleting

Re-suspension

Seeding

Trypsin No trypsin

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3D tissue cultures

No diffusion

Apoptotic death

Secondary necrosis

Necrotic tissue

Diffusion of nutrients and oxygen

Healthy tissue

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Engineering tissuesIn vitro

• Tissue regeneration (tissue proliferation and regeneration on artificial scaffold)

• Bio-artificial organs (liver, skin, pancreas)

In vivo• Blood cell regeneration

(stimulation of stem cell proliferation and differentiation)

• Cell injection into injured tissues (myocardiocytes)

• Large injury regeneration (bio-absorbable scaffolds with or without growth factors)

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Bioreactor• Bioreactors are needed to avoid necrosis

Spinner flask bioreactor

Peristaltic pumps

Drive

Water in

Water out

Air

Counterpressurevalve

Electromagneticvalvefor cooling

Pump

Safety valve

Process Contoller

Heater vessel

Acid BaseAntifoam Substrate

Q

Q valve

Foam

T

pH

pO2

Industrial bioreactor

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Potential uses of cell and tissue replacement therapy• Stroke• Traumatic brain injury• Alzheimer’s disease• Parkinson’s disease• Missing teeth• Wound healing• Bone marrow

transplantation• Spinal cord injury• Osteoarthritis• Rheumatoid arthritis

• Crohn’s disease• Baldness• Blindness• Deafness• Myocardial infarction• Muscular dystrophy• Diabetes• Multisite cancers• Amyotrophic lateral

sclerosis

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Cells in tissue engineeringStem cells• Embryonic• Cord blood• Bone marrow or adipoid tissue derived• Adult, tissue specific Mature differentiated cells• Tissue specific

STEM CELLS (1)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 2

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

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Stem cells• Totipotent• Pluripotent• Multipotent

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Stem cell types

Neural cells

Blood cells

Cultured stem cells

8 cell embryo

Fertilizedoocyte

Totipotent

Blastocyst

Pluripotent

Multipotent

Brain Neural cells Blood cellsBone marrow

Multipotent

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Types of stem cell replications I

Asymmetric, replicating,

differentiating division

Symmetric, differentiating

division

Symmetric, replicating

division

Parental stem cell

Daughter cell Self-reneweddaughter cell

Self-renewal

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Types of stem cell replications IIProliferation capacity

Differentiation capacity

Term

inal

ly d

iffer

entia

ted

cells

Stem cell

Proliferating compartmen

t

Asymmetric divisionAmplification of cell

numbersDifferentiation via progenitor cells

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Sources of Stem Cells• Adult (somatic) stem cells (ASC)• Adult multipotent stem cells• Embryonic stem cells

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Epiblast stem cells (EpiSC) IEpiblast is a tissue that forms at a later stage than ES cells. The epiblasts forms after the developing embryo implants into the uterus.

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Epiblast stem cells (EpiSC) II

Primordialgerm cells (GSC)

Fertilization

Morula

Embryonicstem cells (ESC)

Epiblast

Late blastocyst

EpiSCs

Somatic cell lineages• Endoderm• Mesoderm• Ectoderm

IPS cells

Dedifferentiationand reprogramming

Embryonicgerm cells (EG)

Extraembryonicendoderm (XEN) cells

Primitiveendoderm

Trophoblaststem cells (TS)

Trophectoderm(TE)

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Location of multipotent adult somatic stem cells was identifiedFormerly identified in:

Recently stem cells have been identified

in practically all tissues

BrainSkin

Bone marrow

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Adult (somatic) stem cells (ASC)The microenvironment where stem cells live are called stem cell

niches:Stem cells from fat

Peripheral blood Bone marrow

Skeletal muscle Placenta Hair follicle

Cartilage Bone marrow Cartilage Skeletal muscle Bone marrow SkinMuscle Nerve Tendon Smooth muscle Nerve BrainBone Blood cells Muscle Heart Cartilage Smooth muscleNerve Fat Bone Tendon Fat

Liver Fat Blood vesselsBone marrow CartilageBrain/nerve

BloodHeart

All tissues

ASC in:

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The adult stem cell nicheStem cell niches defined as microenvironments:

Gastrointestinal Brain Bone marrowEsophagus Brain CorneaSmall intestine Nerves RetinaLarge intestine/colon Blood cells PancreasStomach Muscle Liver

All tissues HeartLungSpermatogoniaAmniotic fluidUmbilical cord matrixCord bloodVarious tissues

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Marrow stem cells (MSCs)• Hematopoietic stem cells• Mesenchymal stem cells• Bone Marrow Stromal Stem cells• Endothelial Progenitor cells

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Hematopoietic stem cells (HSC) IThyloLin-Sca-1+

• CD4 and Mac-1 based fractionation resolved three populations and only the Lin-Mac-1-CD4- population was highly enriched for long-term reconstituting HSCs

• c-kit+Sca-1+ThyloFlk-2- long-term repopulating HSC and c-kit+Sca-1+ThyloFlk-2+ short-term repopulating HSC

• Long term repopulating HSCs in mice c-kit+ThyloLin-(Flk-2-) Sca-1+

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Hematopoietic stem cells (HSC) II• In human samples CD34 antigen (function

unknown) is expressed• Additional markers: CD38, CDE90 and CD133• Thy+CD34+ long-term cultures generating

both myeloid and lymphoid lineages• Thy-CD34+ no long-term cultures generating

both myeloid and lymphoid lineages

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Prolymphocyte

Monocyte

Hematopoietic stem cells (HSC) III

Hematopoiesis in Humans Multipotential hematopoietic stem cell(Hemocytoblast)

Myeloblast

Common myeloidprogenitor

Common lymphoidprogenitor

Megakaryoblast

Promegakaryoblast

Megakaryocyte

Thrombocytes

Proerythroblast(Pronormblast)

Basophilic erythroblast

Polychromatic erythroblast

Orthochromatic erythroblast (Normoblast)

Polychromatic erythrocyte (Reticulocyte)

Erythrocyte

Mast cell Lymphoid dendritic cell

Myeloid dendritic cell

Plasma cellMacrophage

Small lymphocyteNatural

killer cell

T lymphocyte B lymphocyte

Lymphoblast

B. promyelocyte

B. myelocyte

B. metamyelocyte

B. band

Basophil

N. promyelocyte

N. myelocyte

N. metamyelocyte

N. band

Neutrophil

E. promyelocyte

E. myelocyte

E. metamyelocyte

E. band

Eosinophil

Monoblast

Promonocyte

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Mesenchymal stem cells (MSC) I• Lacking hematopoietic markers (CD2, CD3,

CD4, CD8, Mac-1/CD11b, CD14, CD15, CD19, CD20, B220, CD45, Thy1 and myeloperoxidase)

• Express: LDL receptor, alkaline phosphatase, smooth muscle actin, type IV collage, laminin, factor VII, MUC18, CD29, CD44, CD49A-F, CD51, CD73, CD105, CD106, CD166+

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Mesenchymal stem cells (MSC) II• MSCs can directly be isolated from bone

marrow based on NGFR (CD271), SSEA-1, SSEA-4, CD140b , CD340 (HER-2), CD349 (Frizzled9)

• Purification is based on: CD29+, CD44+, CD73+, CD105+, CD106+, CD166+ and lack of hematopoietic receptors

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Endothelial progenitor cells (EPC)• Adult EPCs CD34+ or Flk-1+ (VEFR-2)• Additional markers: CD31, Tie2 and E-

selectin, eNOS, LDLR, VEGFR-1,-2

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Bone marrow progenitor cells• HSC and EPC lineages follow similar temporal

and spatial development• HSC and EPC shared markers: VEGFR-2 (Flk-

1), SCL/Tal1, Runx1• Existence of the hemangioblast

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Pericytes SMCs

ECs

Ontogeny of tissue lineages in bone marrow

HSCs Osteoblasts

EPCsMSCs

Stromal Cells

Lymphoid progenitor

s

Myeloid progenitor

sMonocyte

s

T-lineage B-lineage

Meso-AngioblastHemangioblast(Flk-

1+VEcadherin+CD45-)

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Functional interdependency of bone marrow stem cells

HSC

MSC EPC

Array of matrix proteins, adhesion molecules, cytokines is expressed by