© CO

PYRI

GHT U

PM

EXPLORING THE IMMUNOMODULATORY EFFECTS OF HUMAN MESENCHYMAL STEM CELLS ON MONOCYTE FUNCTIONS

MARYAM MAQBOOL

FPSK(p) 2016 15

© CO

PYRI

GHT U

PM

EXPLORING THE IMMUNOMODULATORY EFFECTS OF HUMAN

MESENCHYMAL STEM CELLS ON MONOCYTE FUNCTIONS

By

MARYAM MAQBOOL

Thesis Submitted to the School of Graduates Studies, Universiti Putra Malaysia,

in Fullfilment of the Requirements for the Degree of Doctor of Philosophy

August 2016

© CO

PYRI

GHT U

PM

© CO

PYRI

GHT U

PM

All material contained within the thesis, including without limitation text, logos, icons,

photographs and all other art work, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use maybe made of any material contained within the thesis

for non-commercial purposes from the copyright holder. Commercial use of material

may only be made with the express, prior, written permission of Universiti Putra

Malaysia.

Copyright © Universiti Putra Malaysia

© CO

PYRI

GHT U

PM

To

My

Beloved Mother Dr Ezra Jamal

For her unconditional love, understanding, patience, support

and encouragement that elevated my spirit

© CO

PYRI

GHT U

PM

i

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Doctor of Philosophy

EXPLORING THE IMMUNOMODULATORY EFFECTS OF HUMAN

MESENCHYMAL STEM CELLS ON MONOCYTE FUNCTIONS

By

MARYAM MAQBOOL

August 2016

Chairman : Assoc. Prof. Rajesh Ramasamy, PhD

Faculty : Medicine and Health Sciences

Monocytes are essential phagocytic cells of the innate immune system. They maintain

normal tissue homeostasis but they are also implicated in various chronic

inflammatory diseases. It has been shown that mesenchymal stem cells deliver

immunosuppressive activities on adaptive and innate immune cells. Therefore this

study has explored the less understood immunomodulatory effects of mesenchymal

stem cells on primary and secondary monocyte (cell lines THP-1 and U937) functions.

Primary and secondary monocytes were co-cultured with human umbilical cord-

derived MSCs at appropriate culture conditions to assess the monocyte’s vital

functions such as differentiation, phagocytosis, antigen presentation capability,

cellular proliferation, cell cycle and apoptosis. Based on immunophenotyping and

morphological analysis, mesenchymal stem cells significantly inhibited monocyte

differentiation into dendritic cells and macrophages. Evidenced by lack of expression

of maturation markers, co-stimulatory molecules and MHC class II molecule.

Additionally, the gene expression of selected important genes (TNFRSF11A, TGF-A,

FGFR1 and C3) were analysed using quantitative real time PCR (qPCR) to verify

mesenchymal stem cells mediated inhibition on monocyte’s differentiation at mRNA

level. Mesenchymal stem cells significantly inhibited the expression of TNFRSF11A

and FGFR1 in relevant cells. In the presence of mesenchymal stem cells, monocytes,

dendritic cells and macrophage exhibited declined phagocytosis followed by inability

to stimulate T cell proliferation via PHA antigen presentation. Mesenchymal stem

cells suppressed monocyte proliferation in a dose dependant manner. The anti-

proliferative effect of mesenchymal stem cells was mediated by cell cycle arrest

whereby they were able to arrest monocytes in G0/G1 phase preventing progression

into S and G2/M phases of cell cycle. Cell cycle arrest could potentially lead to cell

apoptosis. However, mesenchymal stem cells significantly enhanced the monocytes

survival and inhibited their apoptosis. Mesenchymal stem cell-mediated

immunosuppression was not confined to primary monocytes it was also extended

towards secondary monocytes. In the presence of mesenchymal stem cells, the

differentiation, proliferation, phagocytosis and apoptosis of secondary monocytes

were significantly abrogated. Over all, this study confers that mesenchymal stem cells

exerted immunosuppressive effects on primary and secondary monocyte functions.

© CO

PYRI

GHT U

PM

ii

Consequently this thesis makes a compelling case for the use of mesenchymal stem

cells in treating and managing the unwanted immune responses such as in graft versus

host disease and other forms of chronic inflammatory diseases. Moving forward it is

imperative to further understand the mechanisms involved in MSC mediated

immunosuppression.

© CO

PYRI

GHT U

PM

iii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

PENEROKAAN KESAN IMMUNOMODULASI SEL INDUK MESENKIMA

MANUSIA PADA FUNGSI MONOSIT

Oleh

MARYAM MAQBOOL

Ogos 2016

Pengerusi : Prof. Madya. Rajesh Ramasamy, PhD

Fakulti : Perubatan dan Sains Kesihatan

Monosit adalah sel fagositik penting dalam sistem imun semulajadi. Monosit

mengekalkan homeostasis tisu normal dan juga dikaitkan dengan pelbagai penyakit

radang kronik. Kajian menunjukkan bahawa sel induk misenkima memberikan

kesan imunorencatan pada sel-sel imun semulajadi dan adaptif. Oleh itu, kajian ini

dijalankan untuk menerokai kesan immunomodulasi misenkima pada fungsi monosit

‘cell line’ THP-1 dan U937. THP-1 dan U937 dikultur bersama misenkima dan

dinilai dari segi fungsi penting monosit seperti pembezaan, fagositosis, keupayaan

persembahan antigen, percambahan sel, kitaran sel dan apoptosis. Berdasarkan

immunophenotyping dan analisis morfologi, misenkima menghalang pembezaan

monosit ke dendritic sel dan makrofaj yang dibuktikan oleh kekurangan ungkapan

penanda kematangan, molekul perangsangan bersama dan molekul MHC kelas II.

Selain itu, ungkapan gen oleh gen penting yang dipilih (TNFRSF11A, TGF-A,

FGFR1 dan C3) telah dikaji dengan menggunakan analisis kuantitatif PCR (qPCR)

untuk mengesahkan perencatan oleh misenkima pada pembezaan monosit di

peringkat mRNA. Pembezaan monosit kepada dendritic sel dan makrofaj

dipendekkan dan juga diiringi dengan pengurangan keupayaan menyampaikan

antigen dan aktiviti fagositik sel-sel yang berkaitan. Dengan kehadiran misenkima,

monosit, dendritic sel dan makrofaj mempamerkan penurunan fungsi fagositosis

diikuti oleh ketidakupayaan untuk merangsang percambahan sel T melalui

persembahan antigen PHA. misenkima menindas percambahan monosit dengan

bergantung kepada dos. Kitaran sel menjadi pengantara kepada kesan anti-

proliferatif misenkima yang mana misenkima memerangkap monosit dalam fasa G0

/ G1, mencegah perkembangan ke S dan G2/M fasa kitaran sel. Penahanan kitaran

sel berpotensi menyebabkan apoptosis sel. Walaubagaimanapun, misenkima

mempertingkatkan kelangsungan hidup monosit dengan ketara dan menghalang

apoptosis monosit. Imunoperencatan misenkima tidak terhad kepada monosit utama

tetapi juga dilanjutkan kepada monosit menengah. Dengan kehadiran misenkima,

pembezaan, perkembangan, fagositosis dan apoptosis monosit menengah ditahan.

Kajian ini menunjukkan bahawa misenkima memberikan kesan imunorencatan

kepada fungsi monosit rendah dan menengah. Oleh itu kajian ini memberikan impak

kepada penggunaan misenkima dalam merawat dan menangani tindakbalas imun

© CO

PYRI

GHT U

PM

iv

yang tidak diingini seperti dalam ‘graft versus host disease’ dan lain-lain penyakit

radang kronik.

© CO

PYRI

GHT U

PM

v

ACKNOWLEDGEMENT

In the name of Allah, the Almighty, the most Gracious and the most Merciful. Praise

is to Allah the cherisher and sustainer of the world. Show us the straightway and O

my Allah! Advance me in knowledge.

Sincere gratitude and appreciation to my supervisor Associate Professor Dr. Rajesh

Ramasamy for his dynamic help and guidance to excel in academic field. He

polished my talents and facilitated me in critical thinking and scientific writing in

order to achieve my goals and accomplish my tasks. My humble gratitude, also goes

to my co-supervisor Professor Dr. Elizabeth George, who gave me her full support

and assistance to complete my research. Special thanks to Dr. Sharmili Vidyadaran

for her help and encouragement with golden ameliorative advice throughout my

study.

Here I would express my sincere appreciation and gratefulness to the members of

Stem Cell and Immunology Laboratory especially my friends Noridzzaida,

Mohadese, Sattar and Zuraidah for their endless help and willingness to share

knowledge and being there throughout my study. Special thanks to the immunology

and stem cell/immunity staff, Mr Izarul, Mr. Anthony, Zura and Marsitah, for

providing technical support and administrative work.

Most importantly I would like to show my gratefulness to my family: my beloved

mother Dr Ezra Jamal, my brother Shah Mohammad Ali my dearest husband Ata Ul

Haq Ansari, my lovely aunty Farhana, my sisters Aeman and Amna for their

unconditional love and prayers. Their endurance and inspiration guided me in

accomplishing my research and writing.

© CO

PYRI

GHT U

PM

© CO

PYRI

GHT U

PM

vii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the Degree of Doctor of Philosophy.

The member of the Supervisory Committee were as follows:

Rajesh Ramasamy, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Elizabeth George, MBBS

Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

Sharmili Vidyadaran,PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduates Studies

Universiti Putra Malaysia

Date:

© CO

PYRI

GHT U

PM

viii

Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other

degree at any other institution;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Pura Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Reseach and Innovation) before thesis is published (in the

form of written, printed or in electronic form) including books, journals,

modules, proceeding, popular writings, seminar papers, manuscripts, posters,

reports, lecture notes, learning modules or any other materials as stated in the

Universiti Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Reseach) Rules 2012. The thesis has undergone plagiarism detection software

Signature: Date:

Name and Matric No: Maryam Maqbool, GS31808

© CO

PYRI

GHT U

PM

ix

Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate Studies)

Rules 2003 (Revision 2012-2013) are adhered to.

Signature:

Name of

Chairman of

Supervisory

Committee: Associate Professor Dr. Rajesh Ramasamy

Signature:

Name of

Member of

Supervisory

Committee: Associate Professor Dr. Sharmili Vidyadaran

Signature:

Name of

Member of

Supervisory

Committee: Professor Dr. Elizabeth George

© CO

PYRI

GHT U

PM

x

TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENT v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xvi

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Immune System 4

2.2 Monocyte 5

2.2.1 Monocyte subsets 6

2.2.2 Plasticity of monocyte 7

2.2.3 Monocyte functions 8

2.2.4 Immune-modulation and therapeutic potential of

monocyte 9

2.3 Dendritic cells 10

2.4 Macrophages 12

2.5 Monocyte cell line 14

2.5.1 THP-1 14

2.5.2 U937 14

2.6 Stimulants 15

2.6.1 LPS 15

2.6.2 PMA 16

2.6.3 IL-3 17

2.6.4 IL-4 17

2.6.5 GM-CSF 17

2.6.6 TNF-A 18

2.7 Quantitative real time PCR (RT-qPCR) 20

2.7.1 Types of real time analysis 21

2.8 Cell cycle 21

2.8.1 Regulation of cell division 21

2.9 Mesenchymal stem cell (MSC) 23

2.9.1 Immunomodulatory activity of MSCs 24

2.9.2 Cellular and molecular interaction of MSCs in innate

immunity 26

2.9.3 Mesenchymal stem cells interaction with

monocyte/macrophage 28

2.9.4 Mesenchymal stem cells interaction with dendritic

cells 29

2.9.5 Therapeutic application of MSCs 32

© CO

PYRI

GHT U

PM

xi

3 MATERIALS AND METHODS / METHODOLOGY

3.1 Cell culture 34

3.2 MSC culture 34

3.3 Monocyte isolation from peripheral human blood 34

3.3.1 Sample 34

3.3.2 Media 34

3.3.3 Monocyte isolation 35

3.3.3.1 Step 1: Isolation of peripheral Blood

mononuclear cells (PBMs) 35

3.3.3.2 Step 2: Antibody and magnetic beads

labelling 36

3.3.3.3 Step 3: Magnetic separation (negative

selection) 37

3.3.3.4 Step 4: Validation of CD14+ monocyte

isolation 37

3.4 Co-culture 39

3.4.1 Differentiation of monocyte, THP-1 and U937 into

dendritic cells and macrophages 39

3.4.1.1 Differentiation of monocyte, THP-1and

U937 into DC 39

3.4.1.2 Differentiation of monocyte, THP-1 and

U937 into macrophage

3.4.2 Characterisation of monocyte, THP-1 and U937

differentiation 41

3.5 Quantitative real time PCR (RT-qPCR)

3.5.1 Isolation of total RNA 41

3.5.2 cDNA synthesis 41

3.5.3 RT-qPCR protocol 41

3.6 Functional assays 42

3.6.1 Phagocytosis 42

3.6.2 Antigen presentation 42

3.6.2.1 T cell isolation 43

3.7 Proliferation assay 44

3.8 Cell cycle assay 45

3.9 Apoptosis assay 45

4 RESULTS 47

4.1 Immunomodulatory effects of MSC on primary monocytes

functions 47

4.1.1 Isolation of human CD14+ monocytes 47

4.1.2 Characterisation of primary monocytes 48

4.1.3 Monocyte differentiate into macrophages dendritic

cells 50

4.1.4 Mesenchymal stem cells inhibit monocyte

differentiation into macrophage and dendritic cells 52

4.1.5 Mesenchymal stem cells effect the gene expression of

monocyte and monocyte-derived DC and macrophage 57

4.1.6 Mesenchymal stem cells inhibit the phagocytosis of

monocyte and monocyte-derived DCs and

macrophages 60

© CO

PYRI

GHT U

PM

xii

4.1.7 Mesenchymal stem cells inhibit the antigen

presenting ability of monocyte, monocyte-derived

DCs and macrophages 62

4.1.8 Mesenchymal stem cells inhibit monocyte

proliferation 67

4.1.9 Mesenchymal stem cells inhibit monocyte cell cycle 72

4.1.10 Mesenchymal stem cells protect monocyte from

apoptosis 75

4.2 Immunomodulatory effects of MSCs on secondary monocyte

THP-1 and U937 78

4.2.1 Characterisation of THP-1 and U937 78

4.2.2 Morphologic assessment of THP-1 and U937

differentiation into DCs and macrophages 79

4.2.3 Mesenchymal stem cells inhibit differentiation of

THP-1 and U937 into macrophage and dendritic cells 82

4.2.4 Mesenchymal stem cells inhibit phagocytosis of THP-

1, U937 and their derived cells 87

4.2.5 Mesenchymal stem cells inhibit THP-1 and U937

proliferation 91

4.2.6 Mesenchymal stem cells inhibit THP-1 and U937 cell

cycle 93

4.2.7 Mesenchymal stem cells inhibit THP-1 and U937

apoptosis 96

5 DISCUSSION 100

5.1 Monocyte Isolation 100

5.2 Mesenchymal stem cells inhibit primary & secondary

monocyte differentiation into dendritic cells and

macrophages 101

5.3 Mesenchymal stem cells prevent the effector functions of

primary & secondary monocyte and derived cells 102

5.4 Mesenchymal stem cells effect primary & secondary

monocyte viability 103

5.5 Mesenchymal stem cells inhibit monocyte proliferation 104

6 CONCLUSION AND RECOMMENDATIONS FOR FUTURE

RESEARCH 106

REFERENCES 107

APPENDICES 125

BIODATA OF STUDENT 130

© CO

PYRI

GHT U

PM

xiii

LIST OF TABLES

Table Page

2.1 Monocyte subset in mice and human blood 7

3.3 Summarised differentiation protocol for monocyte, THP-1 and U937

differentiation into DCs and macrophages 40

4.1 Quantity and quality of monocyte isolation 48

4.2 The impact of MSC on monocyte’s cell cycle 73

A1 Reverse transcription master mix composition 126

A2 PCR reaction mixture 127

A3 PCR experimental program 127

A4 Primer sequence for RT-qPCR 127

© CO

PYRI

GHT U

PM

xiv

LIST OF FIGURES

Figure Page

2.1 Monocyte morphology 6

2.2 Diagrammatic illustration of monocyte plasticity (atherosclerosis) 8

2.3 Diagrammatic illustration of monocyte phagocytosis and antigen

presentation 9

2.4 Dendritic cell morphology 11

2.5 Macrophage morphology 13

2.6 LPS stimulation pathway 16

2.7 GM-CSF regulating DC, monocyte and macrophage

development/function in physiology and pathological conditions 18

2.8 TNF dependant differentiation of monocytes 19

2.9 Overview of the cell cycle phases 22

2.10 Umbilical Cord Mesenchymal stem cell in culture 23

2.11 Schematic illustration of the effects of MSC on immune cells 25

2.12 Mesenchymal stem cells immunosuppression of innate immune

cells 27

2.13 Therapeutic applications of MSC 33

3.1 Ficoll-paque mediated density gradient centrifugation 36

3.2 Indirect magnetic labelling of non-monocytes 36

3.3 Illustration of MACS magnetic separation of unlabelled monocytes 37

3.4 Summarised protocol for monocyte isolation 38

4.1.2 Immunophenotyping of primary monocytes using various myeloid

cell surface markers 49

4.1.3 Morphological assessment of monocyte differentiation 51

4.1.4 The impact of MSC on monocyte differentiation at day 5 53

4.1.4.1 The impact of MSC on monocyte differentiation at day 7 54

4.1.4.2 Immunophenotyping of monocyte differentiation using various

myeloid cell surface markers 56

4.1.5 Evaluation of gene expression by RT-qPCR 59

4.1.6 The impact of MSC on phagocytosis 61

4.1.7 The impact of MSC on antigen presenting property of monocyte

and monocyte derived MAC and DC day 3 64

4.1.7.1 The impact of MSC on antigen presenting property of monocytes

and monocyte derived MAC and DC at day 5 66

4.1.8 Optimisation of monocyte’s proliferation 70

© CO

PYRI

GHT U

PM

xv

4.1.8.1 The impact of MSC on monocyte’s proliferation 71

4.1.9 Flow cytometric representation of monocyte’s cell cycle 74

4.1.10 The impact of MSC on monocyte’s apoptosis 63

4.1.10. 1Flow cytometric representation of monocyte apoptosis at days 5

and 7 74

4.2.1 Immunophenotyping of THP-1 and U937 using various myeloid

cell surface markers 78

4.2.2 Morphological assessment of THP-1 and U937 differentiation 81

4.2.3 The impact of MSC on THP-1 differentiation at day 7 83

4.2.3.1 The impact of MSC on THP-1 differentiation at day 9 84

4.2.3.2 The impact of MSC on U937 differentiation at day 7 85

4.2.3.3 The impact of MSC on U937 differentiation at day 9 86

4.2.4 The impact of MSC on THP-1 and U937 phagocytosis at day 3 88

4.2.4.1 The impact of MSC on phagocytosis of THP-1 derived MAC and

DC at days 3 and 5 89

4.2.4.2 The impact of MSC on phagocytosis of U937 derived DC and

MAC at days 3 and 5 90

4.2.5 The impact of MSC on THP-1 and U937 proliferation 92

4.2.6 The impact of MSC on THP-1 and U937 cell cycle 93

4.2.6.1 Flow cytometric representation of THP-1 and U937 cell cycle at

48hr and 72hr 95

4.2.7 The impact of MSC on THP-1 and U937 apoptosis 95

4.2.7.1 Flow cytometric representation of THP-1 and U937 apoptosis at

48hr and 72hr 99

A1 Immunophenotyping of THP-1 and U937 using various myeloid

cell surface markers 128

A2 Immunophenotyping of THP-1 differentiation using various

myeloid cell cell surface markers 129

© CO

PYRI

GHT U

PM

xvi

LIST OF ABBREVIATIONS

± Plus minus

α Alpha

β Beta

γ Gamma

oC Degree Celsius

L Liter

ml Millilitre

µl Microlitre

g Gram

mg Milligram

µg Microgram

mM Millimolar

µM Micromolar

nM Nanomolar

mg/ml Milligram per millilitre

µg/ml Microgram/millilitre

RT Room temperature

hr Hour

Min Minutes

Sec Seconds

MSC Mesenchymal stem cells

m-DC Monocyte derived DC

m-MAC Monocyte derived MAC

DCs Dendritic cells

mDC Mature dendritic cells

MAC Macrophage

APCs Antigen presenting cells

T cells T lymphocyte

B cells B Lymphocyte

NK cells Natural killer cells

WBC White blood cells

PMA Phorbol-mycitrate-acetate

© CO

PYRI

GHT U

PM

xvii

FMLP N-Formyl -Methionyl-L-Phenylalanine

FPR Formyl peptide receptor

LPS Lipopolysaccharide

LPB Lipopolysaccharide binding protein

TNF-A Tumor necrosis factor alpha

GM-CSF Granulocyte macrophage colony stimulating factor

MAPK Mitogen activated protein kinases

MAPK38 Mitogen activated protein kinases 38

DAG Diacylglicerol

PKC Protein kinase C

PLD Phospholipase D

PLA2 Phospholipase A2

NF-kβ Nuclear factor-kappa beta

O2 Oxygen molecule

PMS Phenazine methosulfate

PS Phosphatidylserine

PI Propidium Iodide

3H-TdR Tritiated thymidine

IF Interferon-gamma

IL-3 Interleukin-3

IL-7 Interleukin-7

IL-8 Interleukin-8

IL-11 Interleukin-11

IL-10 Interleukin-10

IL-6 Interleukin-6

SCF Stem cell factor

SDF-1 Stromal-derived-factor-1

TGFβ1 Transforming growth factors-β1

HGF Hepatocyte growth factor

PGE2 Prostaglandin E2

HO1 Haem oxygenase-1

HLA-G5 Human leukocyte antigen G5

GVHD Graft-versus-host disease

MHC I Major histocompatibility complex class I

MHC II Major histocompatibility complex class II

© CO

PYRI

GHT U

PM

xviii

Fluorescein isothiocyanate

NO2- Nitrite

SD Standard deviation

FBS Fetal bovine serum

NOS Nitric oxide synthase

TLR2 Tol like receptor 2

TLR6 Tol like receptor 6

CD Cluster of differentiation

© CO

PYRI

GHT U

PM

1

CHAPTER 1

INTRODUCTION

The capacity of stem cells to self-renew and give rise to cells of various lineages,

opens an important era of cell-based therapy for various diseases. There are two main

types of stem cells, embryonic and non-embryonic stem cells. Embryonic stem cells

(ESCs) are derived from the inner cell mass of the blastocyst which differentiates into

three germinal layers (ectoderm, mesoderm and endoderm) forming adult organs.

However ethical controversies and occurrence of teratoma obstructed their further

research and clinical usage. On the other hand, non-embryonic stem cells which are

mostly adult stem cells have specialised differentiation potential. They can be isolated

from various tissues and are currently the most commonly used stem cells in

regenerative medicine. Adult stem cells such as mesenchymal stem cells have been

explored as potential therapy for various diseases and have generated immense

interest in the field of regenerative medicine and immune related diseases owing to

their unique biological properties (Kim and Cho, 2013). Mesenchymal stem cells

(MSCs) have generated a great amount of enthusiasm over the past decade as a novel

therapeutic paradigm for a variety of diseases. They have been exploited for their

immunomodulatory properties in the treatment of immune-based disorders, such as

Graft versus Host Disease (GvHD), type 1 diabetes, cardiovascular diseases,

autoimmune disorders and certain type of cancers. Safety and efficacy of using MSCs

as a therapy have recently been demonstrated in various clinical trials (Wei et al.,

2013; Kim and Cho, 2013).

Mesenchymal stem cells are multi-potent progenitor cells that are isolated from the

bone marrow and several other tissues such as adipose tissues, blood, pancreas, dental

pulp, umbilical cord and placenta (Ma et al., 2014). These cells hold remarkable

immunosuppressive properties as shown by inhibiting the proliferation and function

of both, innate and adaptive immune cells. They inhibit proliferation of T and B cells,

natural killer (NK) cells and induce regulatory T cells both in vivo and in vitro. They

modulate the activities of monocytes, dendritic cells (DCs) and macrophages. MSCs

also inhibits neutrophil effector functions and apoptosis (Jiang et al., 2005; Le Blanc

and Davies, 2015; Maqbool et al., 2011; Ramasamy et al., 2008; Rasmusson et al.,

2005). These unique properties make MSCs an ultimate immunosuppressant for

clinical applications. The immunomodulatory effects of MSCs is mediated by a non-

specific, anti-proliferative action of these cells, which is dependent on cell to cell

contact or secreted soluble factors such as indoleamine 2,3-dioxygenase (IDO),

prostaglandin E2 (PGE2), nitric oxide (NO), histocompatibility leucocyte antigen-G

(HLA-G), transforming growth factor (TGF)-β, interferon (IFN)-g and interleukin

(IL)-1b (Ma et al., 2014).

© CO

PYRI

GHT U

PM

2

Monocytes are innate immune cells that provide a first line of immune defence

mechanism in our body and represents 3-10 % of the total white blood cells in adult

humans (Yang et al., 2014). They express various toll-like-receptors (TLR) which

monitors and sense environmental changes. Monocyte are highly plastic and

heterogeneous; can change their functional phenotype in response to environmental

stimulation. As a result they can differentiate into inflammatory and anti-inflammatory

phenotype (Yang et al., 2014). Recruitment of monocytes is essential for effective

control and clearance of viral, bacterial, fungal and protozoal infections. However

additional recruited monocytes can also contribute to the pathogenesis of

inflammatory diseases (Yang et al., 2014; Sheel and Engwerda, 2012). This brings

about to the evident problem statement: The paradoxical functionality of monocytes

which is clearance of infections; at same time contributing towards pathogenesis of

various inflammatory diseases.

Based on the above problem statement the present study is a research model conducted

to assess the immunomodulatory effects of MSCs on primary and secondary (cell lines

THP-1 & U937) monocyte functions in vitro. For this study, primary human

monocytes from peripheral blood and secondary monocyte were utilised. Primary and

secondary monocyte functions such as differentiation into macrophages and dendritic

cells, phagocytosis, antigen presentation, proliferation and cell cycle were explored.

In addition, effects of MSCs on basal monocyte activities such as viability and

apoptosis were also evaluated. This study further investigated the effects of MSCs on

primary monocyte and their derived cells gene expression.

© CO

PYRI

GHT U

PM

3

The general objective of this study is to explore the immunomodulatory effects of

MSCs on primary and secondary monocyte functions.

Therefore the hypotheses of this research are:

1. Mesenchymal stem cells will suppress primary and secondary (cell lines THP-1 & U937) monocytes differentiation towards macrophages and dendritic cells

2. Mesenchymal stem cells will affect the gene expression of primary monocytes and their derived cells.

3. Mesenchymal stem cells will suppress primary and secondary monocytes activities (phagocytosis, antigen presentation, proliferation, cell cycle and

apoptosis).

4. Mesenchymal stem cells will modulate the phagocytic and antigen presenting functions of primary and secondary monocyte and their derived cells

Hence, the objectives of this study are:

1. To investigate the effects of MSCs on primary and secondary (cell lines THP-1 & U937) monocyte differentiation towards macrophages and dendritic cells

2. To decipher the effect of MSCs on primary monocyte and their derived cells via gene expression

3. To evaluate the immunosuppressive effects of MSCs on primary and secondary monocyte activities (phagocytosis, antigen presentation,

proliferation, cell cycle and apoptosis)

4. To assess the effect of MSCs on phagocytic and antigen presenting functions of primary and secondary monocytes and their derived cells

© CO

PYRI

GHT U

PM

© CO

PYRI

GHT U

PM

107

REFERENCES

Abdallah, B., and Kassem, M. (2008). Human mesenchymal stem cells: from basic

biology to clinical applications. Gene Therapy 15, 109-116.

Abdelrazik, H., Spaggiari, G., and Moretta, L. (2010). mesenchymal stem cells inhibit

natural killer cell proliferation, cytotoxicity and cytokine production: role of

indoleamine 2, 3-dioxygenase and prostaglandin E2.: 1746. Transplantation

90, 496.

Ackerman, A. L., and Cresswell, P. (2004). Cellular mechanisms governing cross-

presentation of exogenous antigens. Nature Immunology 5, 678-684.

Aggarwal, S., and Pittenger, M. F. (2005). Human mesenchymal stem cells modulate

allogeneic immune cell responses. Blood 105, 1815-1822.

Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate

immunity. Cell 124, 783-801.

Alam, R. (1998). A brief review of the immune system. Primary Care: Clinics in

Office Practice 25, 727-738.

Aldinucci, A., Rizzetto, L., Pieri, L., Nosi, D., Romagnoli, P., Biagioli, T., Mazzanti,

B., Saccardi, R., Beltrame, L., and Massacesi, L. (2010). Inhibition of immune

synapse by altered dendritic cell actin distribution: a new pathway of

mesenchymal stem cell immune regulation. The Journal of Immunology 185,

5102-5110.

Arnold, C. E., Gordon, P., Barker, R. N., and Wilson, H. M. (2015). The activation

status of human macrophages presenting antigen determines the efficiency of

Th17 responses. Immunobiology 220, 10-19.

Ashhurst, T. M., van Vreden, C., Niewold, P., and King, N. J. C. (2014). The plasticity

of inflammatory monocyte responses to the inflamed central nervous system.

Cellular Immunology 291, 49-57.

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.-J., Pulendran,

B., and Palucka, K. (2000). Immunobiology of dendritic cells. Annual Review

of Immunology 18, 767-811.

Bassi, Ê. J., de Almeida, D. C., Moraes-Vieira, P. M. M., and Câmara, N. O. S. (2012).

Exploring the role of soluble factors associated with immune regulatory

properties of mesenchymal stem cells. Stem Cell Reviews and Reports 8, 329-

342.

Baud, V., and Karin, M. (2001). Signal transduction by tumor necrosis factor and its

relatives. Trends in Cell Biology 11, 372-377.

© CO

PYRI

GHT U

PM

108

Bernardo, M. E., Locatelli, F., and Fibbe, W. E. (2009). Mesenchymal stromal cells.

Annals of the New York Academy of Sciences 1176, 101-117.

Beyth, S., Borovsky, Z., Mevorach, D., Liebergall, M., Gazit, Z., Aslan, H., Galun, E.,

and Rachmilewitz, J. (2005). Human mesenchymal stem cells alter antigen-

presenting cell maturation and induce T-cell unresponsiveness. Blood 105,

2214-2219.

Bhattacharjee, A., Shukla, M., Yakubenko, V. P., Mulya, A., Kundu, S., and Cathcart,

M. K. (2013). IL-4 and IL-13 employ discrete signaling pathways for target

gene expression in alternatively activated monocytes/macrophages. Free

Radical Biology and Medicine 54, 1-16.

Bioimage.usb.edu/immunology/cells-Organs/monocyte.htm

Bingle L., Brown NJ., and Lewis CE. (2002). The role of tumour-associated

macrophages in tumour progression: implications for new anticancer therapies.

Journal Pathology 196, 254–265.

Bitterman, P. B., Saltzman, L. E., Adelberg, S., Ferrans, V. J., and Crystal, R. G.

(1984). Alveolar macrophage replication. One mechanism for the expansion

of the mononuclear phagocyte population in the chronically inflamed lung.

Journal of Clinical Investigation 74, 460.

Biswas SK., Sica A., and Lewis CE. (2008). Plasticity of macrophage function during

tumor progression: regulation by distinct molecular mechanisms. Journal

Immunology 180, 2011–2017.

Bobis, S., Jarocha, D., and Majka, M. (2007). Mesenchymal stem cells: characteristics

and clinical applications. Folia Histochemica et Cytobiologica 44, 215-214.

Baek, Y.-S., Haas, S., Hackstein, H., Bein, G., Hernandez-Santana, M., Lehrach, H.,

Sauer, S., and Seitz, H. (2009). Identification of novel transcriptional

regulators involved in macrophage differentiation and activation in U937 cells.

BMC Immunology 10, 18.

Berges, C., Naujokat, C., Tinapp, S., Wieczorek, H., Höh, A., Sadeghi, M., Opelz, G.,

and Daniel, V. (2005). A cell line model for the differentiation of human

dendritic cells. Biochemical and Biophysical Research Communications 333,

896-907.

Beyth, S., Borovsky, Z., Mevorach, D., Liebergall, M., Gazit, Z., Aslan, H., Galun, E.,

and Rachmilewitz, J. (2005). Human mesenchymal stem cells alter antigen-

presenting cell maturation and induce T-cell unresponsiveness. Blood 105,

2214-2219.

Bohrer, L. R., and Schwertfeger, K. L. (2012). Macrophages promote fibroblast

growth factor receptor-driven tumor cell migration and invasion in a CXCR2-

dependent manner. Molecular Cancer Research 10, 1294-1305.

© CO

PYRI

GHT U

PM

109

Bratton, D. L., Hamid, Q., Boguniewicz, M., Doherty, D. E., Kailey, J., and Leung,

D. (1995). Granulocyte macrophage colony-stimulating factor contributes to

enhanced monocyte survival in chronic atopic dermatitis. Journal of Clinical

Investigation 95, 211.

Brouwer, R. E., van der Hoorn, M., Kluin-Nelemans, H. C., van Zelderen-Bhola, S.,

Willemze, R., and Falkenburg, J. F. (2000). The generation of dendritic-like

cells with increased allostimulatory function from acute myeloid leukemia

cells of various FAB subclasses. Human Immunology 61, 565-574.

Caligiuri, M. A. (2008). Human natural killer cells. Blood 112, 461-469.

Castro-Manrreza, M. E., and Montesinos, J. J. (2015). Immunoregulation by

Mesenchymal Stem Cells: Biological Aspects and Clinical Applications.

Journal of Immunology Research.

Charo, I. F., Myers, S. J., Herman, A., Franci, C., Connolly, A. J., and Coughlin, S. R.

(1994). Molecular cloning and functional expression of two monocyte

chemoattractant protein 1 receptors reveals alternative splicing of the

carboxyl-terminal tails. Proceedings of the National Academy of Sciences 91,

2752-2756.

Chen, X.-D., Akiyama, K., You, Y.-O., Yamaza, T., Chen, C., Tang, L., Jin, Y., and

Shi, S. (2012). Characterization of bone marrow derived mesenchymal stem

cells in suspension. Stem Cell Research Therapy 3, 40.

Cheong, C., Matos, I., Choi, J.-H., Dandamudi, D. B., Shrestha, E., Longhi, M. P.,

Jeffrey, K. L., Anthony, R. M., Kluger, C., and Nchinda, G. (2010). Microbial

stimulation fully differentiates monocytes to DC-SIGN/CD209+ dendritic

cells for immune T cell areas. Cell 143, 416-429.

Chow, A., Brown, B. D., and Merad, M. (2011). Studying the mononuclear phagocyte

system in the molecular age. Nature Reviews Immunology 11, 788-798.

Cohn, L., Chatterjee, B., Esselborn, F., Smed-Sörensen, A., Nakamura, N., Chalouni,

C., Lee, B.-C., Vandlen, R., Keler, T., and Lauer, P. (2013). Antigen delivery

to early endosomes eliminates the superiority of human blood BDCA3+

dendritic cells at cross presentation. The Journal of Experimental Medicine

210, 1049-1063.

Clanchy, F. I., Holloway, A. C., Lari, R., Cameron, P. U., and Hamilton, J. A. (2006).

Detection and properties of the human proliferative monocyte subpopulation.

Journal of Leukocyte Biology 79, 757-766.

Colter, D. C., Class, R., DiGirolamo, C. M., and Prockop, D. J. (2000). Rapid

expansion of recycling stem cells in cultures of plastic-adherent cells from

human bone marrow. Proceedings of the National Academy of Sciences 97,

3213-3218.

© CO

PYRI

GHT U

PM

110

Conti, L., and Gessani, S. (2008). GM-CSF in the generation of dendritic cells from

human blood monocyte precursors: recent advances. Immunobiology 213, 859-

870.

Cutler, A. J., Limbani, V., Girdlestone, J., and Navarrete, C. V. (2010). Umbilical

cord-derived mesenchymal stromal cells modulate monocyte function to

suppress T cell proliferation. The Journal of Immunology 185, 6617-6623.

Davis, I. D., Jefford, M., Parente, P., and Cebon, J. (2003). Rational approaches to

human cancer immunotherapy. Journal of Leukocyte Biology 73, 3-29.

Dale, D. C., Boxer, L., and Liles, W. C. (2008). The phagocytes: neutrophils and

monocytes. Blood 112, 935-945.

Danciger, J. S., Lutz, M., Hama, S., Cruz, D., Castrillo, A., Lazaro, J., Phillips, R.,

Premack, B., and Berliner, J. (2004). Method for large scale isolation, culture

and cryopreservation of human monocytes suitable for chemotaxis, cellular

adhesion assays, macrophage and dendritic cell differentiation. Journal of

Immunological Methods 288, 123-134.

David, J.-P., and Schett, G. (2010). TNF and Bone. Kollias G, Sfikakis PP (eds): TNF

Pathophysiology. Molecular and Cellular Mechanisms 11, 135–144.

Dazzi, F., Ramasamy, R., Glennie, S., Jones, S. P., and Roberts, I. (2006). The role of

mesenchymal stem cells in haemopoiesis. Blood Reviews 20, 161-171.

Dembic, Z. (2000). Immune system protects integrity of tissues. Molecular

Immunology 37, 563-569.

Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P. D., Matteucci,

P., Grisanti, S., and Gianni, A. M. (2002). Human bone marrow stromal cells

suppress T-lymphocyte proliferation induced by cellular or nonspecific

mitogenic stimuli. Blood 99, 3838-3843.

Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D.,

Deans, R., Keating, A., Prockop, D., and Horwitz, E. (2006). Minimal criteria

for defining multipotent mesenchymal stromal cells. The International Society

for Cellular Therapy position statement. Cytotherapy 8, 315-317.

Dreskin, S. C., Thomas, G. W., Dale, S. N., and Heasley, L. E. (2001). Isoforms of

Jun kinase are differentially expressed and activated in human

monocyte/macrophage (THP-1) cells. The Journal of Immunology 166, 5646-

5653.

Du Rocher, B., Mencalha, A. L., Gomes, B. E., and Abdelhay, E. (2012).

Mesenchymal stromal cells impair the differentiation of CD14++ CD16−

CD64+ classical monocytes into CD14++ CD16+ CD64++ activate

monocytes. Cytotherapy 14, 12-25.

© CO

PYRI

GHT U

PM

111

Eggenhofer, E., and Hoogduijn, M. J. (2012). Mesenchymal stem cell-educated

macrophages. Transplant Research 1(12) 1-5.

Eligini, S., Crisci, M., Bono, E., Songia, P., Tremoli, E., Colombo, G. I., and Colli, S.

(2013). Human monocyte‐ derived macrophages spontaneously differentiated in vitro show distinct phenotypes. Journal of Cellular Physiology 228, 1464-

1472.

Ferlazzo, G., Semino, C., Meta, M., Procopio, F., Morandi, B., and Melioli, G.

(2002a). T lymphocytes express B7 family molecules following interaction

with dendritic cells and acquire bystander costimulatory properties. European

Journal of Immunology 32, 3092-3101.

Ferlazzo, G., Tsang, M. L., Moretta, L., Melioli, G., Steinman, R. M., and Münz, C.

(2002b). Human dendritic cells activate resting natural killer (NK) cells and

are recognized via the NKp30 receptor by activated NK cells. The Journal of

Experimental Medicine 195, 343-351.

François, M., Romieu-Mourez, R., Li, M., and Galipeau, J. (2012). Human MSC

suppression correlates with cytokine induction of indoleamine 2, 3-

dioxygenase and bystander M2 macrophage differentiation. Molecular

Therapy 20, 187-195.

Friedenstein, A., Chailakhjan, R., and Lalykina, K. (1970). The development of

fibroblast colonies in monolayer cultures of guinea‐ pig bone marrow and spleen cells. Cell Proliferation 3, 393-403.

Friedenstein, A. J., Gorskaja, J., and Kulagina, N. (1976). Fibroblast precursors in

normal and irradiated mouse hematopoietic organs. Experimental Hematology

4, 267-274.

Ganz, T. (1999). Defensins and host defense. Science 286, 420-421.

Geissmann, F., Gordon, S., Hume, D. A., Mowat, A. M., and Randolph, G. J. (2010).

Unravelling mononuclear phagocyte heterogeneity. Nature Reviews

Immunology 10, 453-460.

Geissmann, F., Jung, S., and Littman, D. R. (2003). Blood monocytes consist of two

principal subsets with distinct migratory properties. Immunity 19, 71-82.

Geissmann F., Manz MG., Jung S., Sieweke MH., and Merad M, et al. (2010).

Development of monocytes, macrophages, and dendritic cells. Science 327:

656 – 661.

Gerard, C., and Rollins, B. J. (2001). Chemokines and disease. Nature Immunology 2,

108-115.

Glenn, J. D., and Whartenby, K. A. (2014). Mesenchymal stem cells: Emerging

mechanisms of immunomodulation and therapy. World Journal of Stem Cells

6 (5), 526-539.

© CO

PYRI

GHT U

PM

112

Goldszmid, R. S., Caspar, P., Rivollier, A., White, S., Dzutsev, A., Hieny, S., Kelsall,

B., Trinchieri, G., and Sher, A. (2012). NK cell-derived interferon-γ

orchestrates cellular dynamics and the differentiation of monocytes into

dendritic cells at the site of infection. Immunity 36, 1047-1059.

Goncalves, R., Zhang, X., Cohen, H., Debrabant, A., and Mosser, D. M. (2011).

Platelet activation attracts a subpopulation of effector monocytes to sites of

Leishmania major infection. The Journal of Experimental Medicine 208, 1253-

1265.

Gordon, S., and Martinez, F. O. (2010). Alternative activation of macrophages:

mechanism and functions. Immunity 32, 593-604.

Gordon, S., and Taylor, P. R. (2005). Monocyte and macrophage heterogeneity.

Nature Reviews Immunology 5, 953-964.

Guha, M., and Mackman, N. (2001). LPS induction of gene expression in human

monocytes. Cellular Signalling 13, 85-94.

Guilliams, M., Ginhoux, F., Jakubzick, C., Naik, S. H., Onai, N., Schraml, B. U.,

Segura, E., Tussiwand, R., and Yona, S. (2014). Dendritic cells, monocytes

and macrophages: a unified nomenclature based on ontogeny. Nature Reviews

Immunology 14, 571-578.

Guthridge, M. A., Stomski, F. C., Thomas, D., Woodcock, J. M., Bagley, C. J., Berndt,

M. C., and Lopez, A. F. (1998). Mechanism of Activation of the GM‐ CSF, IL‐ 3, and IL‐ 5 Family of Receptors. Stem Cells 16, 301-313.

Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996). Real time

quantitative PCR. Genome Research 6, 986-994.

Henderson, B., Glynn, L., Bitensky, L., and Chayen, J. (1981). Evidence for cell

division in synoviocytes in acutely inflamed rabbit joints. Annals of the

Rheumatic Diseases 40, 177-181.

Hercus, T. R., Thomas, D., Guthridge, M. A., Ekert, P. G., King-Scott, J., Parker, M.

W., and Lopez, A. F. (2009). The granulocyte-macrophage colony-stimulating

factor receptor: linking its structure to cell signaling and its role in disease.

Blood 114, 1289-1298.

Heusinkveld M., De Vos Van Steenwijk PJ., Goedemans R., Ramwadhdoebe TH.,

Gorter A, et al. (2011). M2 macrophages induced by prostaglandin E2 and IL-

6 from cervical carcinoma are switched to activated M1 macrophages by CD4+

Th1 cells. Journal Immunology 187, 1157–1165.

Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). Kinetic PCR analysis:

real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026-

1030.

© CO

PYRI

GHT U

PM

113

Hoebe, K., Janssen, E., and Beutler, B. (2004). The interface between innate and

adaptive immunity. Nature Immunology 5, 971-974.

Hoeffel, G., and Ginhoux, F. (2015). Ontogeny of tissue-resident macrophages.

Frontiers in Immunology 6, 486.

Hubo M., Trinschek B., Kryczanowsky F., Tuettenberg A., Helmut and Jonuleit.

(2013). Frontier of Immunology 4 (82) 1-14.

Iwamoto, S., Iwai, S.-i., Tsujiyama, K., Kurahashi, C., Takeshita, K., Naoe, M.,

Masunaga, A., Ogawa, Y., Oguchi, K., and Miyazaki, A. (2007). TNF-α drives

human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking

Th1 and Th17 responses. The Journal of Immunology 179, 1449-1457.

Iwasaki, A. (2007). Division of labor by dendritic cells. Cell 128, 435-436.

Jenkins, S. J., Ruckerl, D., Cook, P. C., Jones, L. H., Finkelman, F. D., van Rooijen,

N., MacDonald, A. S., and Allen, J. E. (2011). Local macrophage proliferation,

rather than recruitment from the blood, is a signature of TH2 inflammation.

Science 332, 1284-1288.

Jonuleit, H., Schmitt, E., Schuler, G., Knop, J., and Enk, A. H. (2000). Induction of

interleukin 10–producing, nonproliferating CD4+ T cells with regulatory

properties by repetitive stimulation with allogeneic immature human dendritic

cells. The Journal of Experimental Medicine 192, 1213-1222.

Jiang, X.-X., Zhang, Y., Liu, B., Zhang, S.-X., Wu, Y., Yu, X.-D., and Mao, N. (2005).

Human mesenchymal stem cells inhibit differentiation and function of

monocyte-derived dendritic cells. Blood 105, 4120-4126.

Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-

Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., and Blackstad, M. (2002).

Pluripotency of mesenchymal stem cells derived from adult marrow. Nature

418, 41-49.

Johnson, D., and Walker, C. (1999). Cyclins and cell cycle checkpoints. Annual review

of Pharmacology and Toxicology 39, 295-312.

Karantalis, V., and Hare, J. M. (2015). Use of mesenchymal stem cells for therapy of

cardiac disease. Circulation Research 116, 1413-1430.

Kassem, M., and Abdallah, B. M. (2008). Human bone-marrow-derived mesenchymal

stem cells: biological characteristics and potential role in therapy of

degenerative diseases. Cell and Tissue Research 331, 157-163.

Keneth Todar, University of Wisconsin, Department of Bacteriology

Kawai, T., and Akira, S. (2011). Toll-like receptors and their crosstalk with other

innate receptors in infection and immunity. Immunity 34, 637-650.

© CO

PYRI

GHT U

PM

114

Kim, J., and Hematti, P. (2009). Mesenchymal stem cell–educated macrophages: A

novel type of alternatively activated macrophages. Experimental Hematology

37, 1445-1453.

Kim, N., and Cho, S.-G. (2013). Clinical applications of mesenchymal stem cells. The

Korean Journal of Internal Medicine 28, 387-402.

Kim, S. W., Choi, S. M., Choo, Y. S., Kim, I. K., Song, B. W., and Kim, H. S. (2015).

Flt3 Ligand Induces Monocyte Proliferation and Enhances the Function of

Monocyte‐ Derived Dendritic Cells In Vitro. Journal of Cellular Physiology 230, 1740-1749.

Koç, O. N., Gerson, S. L., Cooper, B. W., Dyhouse, S. M., Haynesworth, S. E., Caplan,

A. I., and Lazarus, H. M. (2000). Rapid hematopoietic recovery after

coinfusion of autologous-blood stem cells and culture-expanded marrow

mesenchymal stem cells in advanced breast cancer patients receiving high-

dose chemotherapy. Journal of Clinical Oncology 18, 307-307.

Kolf, C. M., Cho, E., and Tuan, R. S. (2007). Biology of adult mesenchymal stem

cells: regulation of niche, self-renewal and differentiation. Arthritis Research

Therapy 9 (204), 1-10.

Kotobuki, N., Ioku, K., Kawagoe, D., Fujimori, H., Goto, S., and Ohgushi, H. (2005).

Observation of osteogenic differentiation cascade of living mesenchymal stem

cells on transparent hydroxyapatite ceramics. Biomaterials 26, 779-785.

Krampera, M., Franchini, M., Pizzolo, G., and Aprili, G. (2007a). Mesenchymal stem

cells: from biology to clinical use. Blood Transfusion 5, 120-129.

Krampera, M., Marconi, S., Pasini, A., Galiè, M., Rigotti, G., Mosna, F., Tinelli, M.,

Lovato, L., Anghileri, E., and Andreini, A. (2007b). Induction of neural-like

differentiation in human mesenchymal stem cells derived from bone marrow,

fat, spleen and thymus. Bone 40, 382-390.

Langstein, J., Michel, J., and Schwarz, H. (1999). CD137 induces proliferation and

endomitosis in monocytes. Blood 94, 3161-3168.

Langstein, J., and Schwarz, H. (1999). Identification of CD137 as a potent monocyte

survival factor. Journal of Leukocyte Biology 65, 829-833.

Laranjeira, P., Gomes, J., Pedreiro, S., Pedrosa, M., Martinho, A., Antunes, B.,

Ribeiro, T., Santos, F., Domingues, R., and Abecasis, M. (2015). Human Bone

Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine

Production by Peripheral Blood Monocytes Subpopulations and Myeloid

Dendritic Cells. Stem Cells International ID 819084, 1-15.

Le Naour, F., Hohenkirk, L., Grolleau, A., Misek, D. E., Lescure, P., Geiger, J. D.,

Hanash, S., and Beretta, L. (2001). Profiling changes in gene expression during

differentiation and maturation of monocyte-derived dendritic cells using both

© CO

PYRI

GHT U

PM

115

oligonucleotide microarrays and proteomics. Journal of Biological Chemistry

276, 17920-17931.

Lehtonen, A., Ahlfors, H., Veckman, V., Miettinen, M., Lahesmaa, R., and Julkunen,

I. (2007). Gene expression profiling during differentiation of human

monocytes to macrophages or dendritic cells. Journal of Leukocyte Biology 82,

710-720.

Le Blanc, K., and Davies, L. C. (2015). Mesenchymal stromal cells and the innate

immune response. Immunology letters 168, 140-146.

Le Blanc, K., and Ringdén, O. (2005). Immunobiology of human mesenchymal stem

cells and future use in hematopoietic stem cell transplantation. Biology of

Blood and Marrow Transplantation 11, 321-334.

Lea, N. C., Orr, S. J., Stoeber, K., Williams, G. H., Lam, E. W.-F., Ibrahim, M. A.,

Mufti, G. J., and Thomas, N. S. B. (2003). Commitment point during G0→ G1

that controls entry into the cell cycle. Molecular and Cellular Biology 23,

2351-2361.

León, B., and Ardavín, C. (2008). Monocyte-derived dendritic cells in innate and

adaptive immunity. Immunology and Cell Biology 86, 320-324.

León, B., López-Bravo, M., and Ardavín, C. (2007). Monocyte-derived dendritic cells

formed at the infection site control the induction of protective T helper 1

responses against Leishmania. Immunity 26, 519-531.

Li, Y.-P., Paczesny, S., Lauret, E., Poirault, S., Bordigoni, P., Mekhloufi, F., Hequet,

O., Bertrand, Y., Ou-Yang, J.-P., and Stoltz, J.-F. (2008). Human

mesenchymal stem cells license adult CD34+ hemopoietic progenitor cells to

differentiate into regulatory dendritic cells through activation of the Notch

pathway. The Journal of Immunology 180, 1598-1608.

Lindstedt, M., Johansson‐ Lindbom, B., and Borrebaeck, C. A. (2002). Global reprogramming of dendritic cells in response to a concerted action of

inflammatory mediators. International Immunology 14, 1203-1213.

Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data

using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25, 402-

408.

Locatelli, F., Pende, D., Maccario, R., Mingari, M. C., Moretta, A., and Moretta, L.

(2009). Haploidentical hemopoietic stem cell transplantation for the treatment

of high-risk leukemias: how NK cells make the difference. Clinical

Immunology 133, 171-178.

Marks, S. (1983). The origin of osteoclasts. Journal of Oral Pathology & Medicine

12, 226-256.

© CO

PYRI

GHT U

PM

116

Ma, S., Xie, N., Li, W., Yuan, B., Shi, Y., and Wang, Y. (2014). Immunobiology of

mesenchymal stem cells. Cell Death & Differentiation 21, 216-225.

Maggini, J., Mirkin, G., Bognanni, I., Holmberg, J., Piazzón, I. M., Nepomnaschy, I.,

Costa, H., Cañones, C., Raiden, S., and Vermeulen, M. (2010). Mouse bone

marrow-derived mesenchymal stromal cells turn activated macrophages into a

regulatory-like profile. PloS one 5, e9252.

Malinen, E., Kassinen, A., Rinttilä, T., and Palva, A. (2003). Comparison of real-time

PCR with SYBR Green I or 5′-nuclease assays and dot-blot hybridization with

rDNA-targeted oligonucleotide probes in quantification of selected faecal

bacteria. Microbiology 149, 269-277.

Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., and Locati, M. (2004).

The chemokine system in diverse forms of macrophage activation and

polarization. Trends In Immunology 25, 677-686.

Maqbool, M., Vidyadaran, S., George, E., and Ramasamy, R. (2011). Human

mesenchymal stem cells protect neutrophils from serum‐ deprived cell death. Cell Biology International 35, 1247-1251.

Martinez-Pomares, L., and Gordon, S. (2007). Antigen presentation the macrophage

way. Cell 131, 641-643.

Martinez FO., Sica A., Mantovani A., and Locati M (2008). Macrophage activation

and polarization. Frontier Biosciences 13, 453–461.

McInnes, I. B., and Schett, G. (2007). Cytokines in the pathogenesis of rheumatoid

arthritis. Nature Reviews Immunology 7, 429-442.

Medzhitov, R., and Janeway, C. A. (2002). Decoding the patterns of self and nonself

by the innate immune system. Science 296, 298-300.

Melief, S. M., Geutskens, S. B., Fibbe, W. E., and Roelofs, H. (2013). Multipotent

stromal cells skew monocytes towards an anti-inflammatory interleukin-10-

producing phenotype by production of interleukin-6. Haematologica 98, 888-

895.

Mehta NN.,and Reilly MP. (2012). Monocyte Mayhem: Do Subtypes Modulate

Distinct Atherosclerosis Phenotypes? Circulatory Cardiovasccular Genetics

5, 7-9.

Merad, M., Sathe, P., Helft, J., Miller, J., and Mortha, A. (2013). The dendritic cell

lineage: ontogeny and function of dendritic cells and their subsets in the steady

state and the inflamed setting. Annual Review of Immunology 31.

Minafra, L., Di Cara, G., Albanese, N. N., and Cancemi, P. (2011). Proteomic

differentiation pattern in the U937 cell line. Leukemia research 35, 226-236.

© CO

PYRI

GHT U

PM

117

Miska, E. A. (2005). How microRNAs control cell division, differentiation and death.

Current Opinion in Genetics & Development 15, 563-568.

Mitra, S. K., and Schlaepfer, D. D. (2006). Integrin-regulated FAK–Src signaling in

normal and cancer cells. Current Opinion in Cell Biology 18, 516-523.

Moretta, A. (2002). Natural killer cells and dendritic cells: rendezvous in abused

tissues. Nature Reviews Immunology 2, 957-965.

Moretta, L., Ferlazzo, G., Bottino, C., Vitale, M., Pende, D., Mingari, M. C., and

Moretta, A. (2006). Effector and regulatory events during natural killer–

dendritic cell interactions. Immunological Reviews 214, 219-228.

Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998). Quantification of low-copy

transcripts by continuous SYBR Green I monitoring during amplification.

Biotechniques 24, 954-8, 960, 962.

Moser, M., and Murphy, K. M. (2000). Dendritic cell regulation of TH1-TH2

development. Nature Immunology 1, 199-205.

Mosser, D. M., and Edwards, J. P. (2008). Exploring the full spectrum of macrophage

activation. Nature Reviews Immunology 8, 958-969.

Murphy, M. B., Moncivais, K., and Caplan, A. I. (2013). Mesenchymal stem cells:

environmentally responsive therapeutics for regenerative medicine.

Experimental & Molecular Medicine 45, e54.

Murray, P. J., and Wynn, T. A. (2011a). Obstacles and opportunities for understanding

macrophage polarization. Journal of Leukocyte Biology 89, 557-563.

Murray, P. J., and Wynn, T. A. (2011b). Protective and pathogenic functions of

macrophage subsets. Nature Reviews Immunology 11, 723-737.

Nauta, A. J., Kruisselbrink, A. B., Lurvink, E., Willemze, R., and Fibbe, W. E. (2006).

Mesenchymal stem cells inhibit generation and function of both CD34+-

derived and monocyte-derived dendritic cells. The Journal of Immunology

177, 2080-2087.

Nelms k., Keegan DA., Zamorano J., Ryan JJ., and Paul EW.(1999). The IL-4

Receptor: Signaling Mechanisms and Biologic Functions. Annual Review of

Immunology 17, 701-738.

Nuttall, G. H. (2015). "Blood immunity and blood relationship," Cambridge

University Press, Chapter 1 page 17.

Palmer, S., Wiegand, A. P., Maldarelli, F., Bazmi, H., Mican, J. M., Polis, M., Dewar,

R. L., Planta, A., Liu, S., and Metcalf, J. A. (2003). New real-time reverse

transcriptase-initiated PCR assay with single-copy sensitivity for human

immunodeficiency virus type 1 RNA in plasma. Journal of Clinical

Microbiology 41, 4531-4536.

© CO

PYRI

GHT U

PM

118

Parkin, J., and Cohen, B. (2001). An overview of the immune system. The Lancet 357,

1777-1789.

Passmore, J., Lukey, P., and Ress, S. (2001). The human macrophage cell line U937

as an in vitro model for selective evaluation of mycobacterial antigen‐ specific cytotoxic T‐ cell function. Immunology 102, 146-156.

Perkins, D. J., Were, T., Davenport, G. C., Kempaiah, P., Hittner, J. B., and Ong'echa,

J. M. (2011). Severe malarial anemia: innate immunity and pathogenesis.

International Journal of biological Sciences 7, 1427.

Perera, L. P., and Waldmann, T. A. (1998). Activation of human monocytes induces

differential resistance to apoptosis with rapid down regulation of caspase-

8/FLICE. Proceedings of the National Academy of Sciences 95, 14308-14313.

Petite, H., Viateau, V., Bensaid, W., Meunier, A., de Pollak, C., Bourguignon, M.,

Oudina, K., Sedel, L., and Guillemin, G. (2000). Tissue-engineered bone

regeneration. Nature Biotechnology 18, 959-963.

Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J.

D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999).

Multilineage potential of adult human mesenchymal stem cells. Science 284,

143-147.

Porcheray F., Viaud S., Rimaniol AC., Leone C., Samah B, et al. (2005). Macrophage

activation switching: an asset for the resolution of inflammation. Clinical

Experimental Immunology 142, 481–489.

Pulendran, B., Banchereau, J., Burkeholder, S., Kraus, E., Guinet, E., Chalouni, C.,

Caron, D., Maliszewski, C., Davoust, J., and Fay, J. (2000). Flt3-ligand and

granulocyte colony-stimulating factor mobilize distinct human dendritic cell

subsets in vivo. The Journal of Immunology 165, 566-572.

Qian BZ., and Pollard JW. (2010). Macrophage diversity enhances tumor progression

and metastasis. Cell, 141: 39–51.

Qin, Z. (2012). The use of THP-1 cells as a model for mimicking the function and

regulation of monocytes and macrophages in the vasculature. Atherosclerosis

221, 2-11.

Qu, C., Brinck-Jensen, N.-S., Zang, M., and Chen, K. (2014). Monocyte-derived

dendritic cells: targets as potent antigen-presenting cells for the design of

vaccines against infectious diseases. International Journal of Infectious

Diseases 19, 1-5.

Qu, C., Moran, T. M., and Randolph, G. J. (2003). Autocrine type I IFN and contact

with endothelium promote the presentation of influenza A virus by monocyte-

derived APC. The Journal of Immunology 170, 1010-1018.

© CO

PYRI

GHT U

PM

119

Qu, C., Nguyen, V. A., Merad, M., and Randolph, G. J. (2009). MHC class I/peptide

transfer between dendritic cells overcomes poor cross-presentation by

monocyte-derived APCs that engulf dying cells. The Journal of Immunology

182, 3650-3659.

Raffaghello, L., Bianchi, G., Bertolotto, M., Montecucco, F., Busca, A., Dallegri, F.,

Ottonello, L., and Pistoia, V. (2008). Human mesenchymal stem cells inhibit

neutrophil apoptosis: a model for neutrophil preservation in the bone marrow

niche. Stem Cells 26, 151-162.

Rahimian, A., Soleimani, M., Kaviani, S., Aghaee-Bakhtiari, S., Atashi, A., Arefian,

E., and Nikougoftar, M. (2011). Bypassing the maturation arrest in myeloid

cell line U937 by over-expression of microRNA-424. Hematology 16, 298-

302.

Ramasamy, R., Tong, C. K., Seow, H. F., Vidyadaran, S., and Dazzi, F. (2008). The

immunosuppressive effects of human bone marrow-derived mesenchymal

stem cells target T cell proliferation but not its effector function. Cellular

Immunology 251, 131-136.

Ramasamy, R., Fazekasova, H., Lam, E. W.-F., Soeiro, I., Lombardi, G., and Dazzi,

F. (2007a). Mesenchymal stem cells inhibit dendritic cell differentiation and

function by preventing entry into the cell cycle. Transplantation 83, 71-76.

Ramasamy, R., Lam, E. W., Soeiro, I., Tisato, V., Bonnet, D., and Dazzi, F. (2007b).

Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells:

impact on in vivo tumor growth. Leukemia 21, 304-310.

Randolph, G. J., Jakubzick, C., and Qu, C. (2008). Antigen presentation by monocytes

and monocyte-derived cells. Current Opinion in Immunology 20, 52-60.

Rasaiyaah, J., Yong, K., Katz, D. R., Kellam, P., and Chain, B. M. (2007). Dendritic

cells and myeloid leukaemias: plasticity and commitment in cell

differentiation. British Journal of Haematology 138, 281-290.

Rasmusson, I., Ringdén, O., Sundberg, B., and Le Blanc, K. (2005). Mesenchymal

stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by

different mechanisms. Experimental Cell Research 305, 33-41.

Ripa, R. S., Haack-Sørensen, M., Wang, Y., Jørgensen, E., Mortensen, S., Bindslev,

L., Friis, T., and Kastrup, J. (2007). Bone Marrow–Derived Mesenchymal Cell

Mobilization by Granulocyte-Colony Stimulating Factor After Acute

Myocardial Infarction Results From the Stem Cells in Myocardial Infarction

(STEMMI) Trial. Circulation 116, I-24-I-30.

Rivollier, A., He, J., Kole, A., Valatas, V., and Kelsall, B. L. (2012). Inflammation

switches the differentiation program of Ly6Chi monocytes from

antiinflammatory macrophages to inflammatory dendritic cells in the colon.

The Journal of Experimental Medicine 209, 139-155.

© CO

PYRI

GHT U

PM

120

Rocher, B. D., Mencalha, A. L., Gomes, B. E., and Abdelhay, E. (2012). Mesenchymal

stromal cells impair the differentiation of CD14++ CD16− CD64+ classical

monocytes into CD14++ CD16+ CD64++ activate monocytes. Cytotherapy

14, 12-25.

Saha, P., and Geissmann, F. (2011). Toward a functional characterization of blood

monocytes. Immunology and Cell Biology 89, 2-4.

Santegoets, S. J., van den Eertwegh, A. J., van de Loosdrecht, A. A., Scheper, R. J.,

and de Gruijl, T. D. (2008). Human dendritic cell line models for DC

differentiation and clinical DC vaccination studies. Journal of Leukocyte

Biology 84, 1364-1373.

Shi, C., and Pamer, E. G. (2011). Monocyte recruitment during infection and

inflammation. Nature Reviews Immunology 11, 762-774.

Silveira, G. F., Wowk, P. F., Machado, A. M. B., dos Santos, C. N. D., and Bordignon,

J. (2013). Immature dendritic cells generated from cryopreserved human

monocytes show impaired ability to respond to LPS and to induce allogeneic

lymphocyte proliferation. PloS one 8, 9 e71291.

Schmittgen, T. D., and Zakrajsek, B. A. (2000). Effect of experimental treatment on

housekeeping gene expression: validation by real-time, quantitative RT-PCR.

Journal of Biochemical and Biophysical Methods 46, 69-81.

Sekiya, I., Larson, B. L., Smith, J. R., Pochampally, R., Cui, J. G., and Prockop, D. J.

(2002). Expansion of human adult stem cells from bone marrow stroma:

conditions that maximize the yields of early progenitors and evaluate their

quality. Stem Cells 20, 530-541.

Serbina, N. V., Jia, T., Hohl, T. M., and Pamer, E. G. (2008). Monocyte-mediated

defense against microbial pathogens. Annual Review of Immunology 26, 421.

Serbina, N. V., and Pamer, E. G. (2006). Monocyte emigration from bone marrow

during bacterial infection requires signals mediated by chemokine receptor

CCR2. Nature Immunology 7, 311-317.

Serbina, N. V., Salazar-Mather, T. P., Biron, C. A., Kuziel, W. A., and Pamer, E. G.

(2003). TNF/iNOS-producing dendritic cells mediate innate immune defense

against bacterial infection. Immunity 19, 59-70.

Sheel, M., and Engwerda, C. R. (2012). The diverse roles of monocytes in

inflammation caused by protozoan parasitic diseases. Trends in Parasitology

28, 408-416.

Shi, M., Liu, Z. W., and Wang, F. S. (2011). Immunomodulatory properties and

therapeutic application of mesenchymal stem cells. Clinical & Experimental

Immunology 164, 1-8.

© CO

PYRI

GHT U

PM

121

Silva, W. A., Covas, D. T., Panepucci, R. A., Proto‐ Siqueira, R., Siufi, J. L., Zanette, D. L., Santos, A. R., and Zago, M. A. (2003). The profile of gene expression

of human marrow mesenchymal stem cells. Stem Cells 21, 661-669.

Song, J., Cong, S., Li, Y., Bai, L., and Cao, G. (2015). [Human amniotic mesenchymal

stem cells inhibit allogeneic lymphocyte proliferation and reduce the secretion

of interferon γ]. Xi bao yu fen zi mian yi xue za zhi= Chinese Journal of

Cellular and Molecular Immunology 31, 333-337.

Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N., and Papamichail,

M. (2006). Characterization of the optimal culture conditions for clinical scale

production of human mesenchymal stem cells. Stem Cells 24, 462-471.

Spaggiari, G., Abdelrazik, H., Becchetti, F., and Moretta, L. (2009). Mesenchymal

stem cells inhibit dendritic cell maturation and function by selectively

interfering with the generation of immature DCs: central role of MSC-derived

prostaglandin E2. Blood 113, 6576-6583.

Spaggiari, G. M., Capobianco, A., Becchetti, S., Mingari, M. C., and Moretta, L.

(2006). Mesenchymal stem cell-natural killer cell interactions: evidence that

activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-

2-induced NK-cell proliferation. Blood 107, 1484-1490.

Spaggiari, G. M., and Moretta, L. (2013). Cellular and molecular interactions of

mesenchymal stem cells in innate immunity. Immunology and Cell Biology 91,

27-31.

Sreekumar, T., Ansari, M., Chandra, G., and Sharma, T. (2014). Isolation and

characterization of Buffalo Wharton’s Jelly derived mesenchymal stem cells.

Journal Stem Cell Research Therapy 4, 1-6.

Steinert, A. F., Rackwitz, L., Gilbert, F., Nöth, U., and Tuan, R. S. (2012). Concise

review: the clinical application of mesenchymal stem cells for musculoskeletal

regeneration: current status and perspectives. Stem Cells Translational

Medicine 1, 237-247.

Swirski, F. K., Nahrendorf, M., Etzrodt, M., Wildgruber, M., Cortez-Retamozo, V.,

Panizzi, P., Figueiredo, J.-L., Kohler, R. H., Chudnovskiy, A., and Waterman,

P. (2009). Identification of splenic reservoir monocytes and their deployment

to inflammatory sites. Science 325, 612-616.

Tada, M., Ichiishi, E., Saito, R., Emoto, N., Niwano, Y., and Kohno, M. (2009).

Myristic acid, a side chain of phorbol myristate acetate (PMA), can activate

human polymorphonuclear leukocytes to produce oxygen radicals more

potently than PMA. Journal of Clinical Biochemistry and Nutrition 45, 309.

Tahara, E., Kadara, H., Lacroix, L., Lotan, D., and Lotan, R. (2009). Activation of

protein kinase C by phorbol 12-myristate 13-acetate suppresses the growth of

lung cancer cells through KLF6 induction. Cancer Biology & Therapy 8, 801-

807.

© CO

PYRI

GHT U

PM

122

Tormo1 VR., Company C., Rodríguez-Ubreva1 J., Rica1 de la L., Urquiza1 MJ., Biola

MJ., et al. (2016). IL-4 orchestrates STAT6-mediated DNA demethylation

leading to dendritic cell differentiation. Genome Biology 17(4), 2-18.

Trivedi, H., and Vanikar, A. (2013). Mesenchymal stem cells and solid organ

transplantation. Cell R4 1, 123-136.

Tsuchiya, S., Kobayashi, Y., Goto, Y., Okumura, H., Nakae, S., Konno, T., and Tada,

K. (1982). Induction of maturation in cultured human monocytic leukemia

cells by a phorbol diester. Cancer Research 42, 1530-1536.

Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K.

(1980). Establishment and characterization of a human acute monocytic

leukemia cell line (THP‐ 1). International Journal of Cancer 26, 171-176.

Tuan, R. S., Boland, G., and Tuli, R. (2003). Adult mesenchymal stem cells and cell-

based tissue engineering. Arthritis Research and Therapy 5, 32-45.

Tung, J.-P., Fraser, J. F., Wood, P., and Fung, Y. L. (2009). Respiratory burst function

of ovine neutrophils. BMC Immunology 10, 25.

Uccelli, A., Moretta, L., and Pistoia, V. (2008). Mesenchymal stem cells in health and

disease. Nature Reviews Immunology 8, 726-736.

Undale, A. H., Westendorf, J. J., Yaszemski, M. J., and Khosla, S. (2009).

Mesenchymal stem cells for bone repair and metabolic bone diseases. In

"Mayo Clinic Proceedings", Vol. 84, pp. 893-902. Elsevier.

Van Furth, R., and Cohn, Z. A. (1968). The origin and kinetics of mononuclear

phagocytes. The Journal of Experimental Medicine 128, 415-435.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A.,

and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-

PCR data by geometric averaging of multiple internal control genes. Genome

Biology 3(7).

Vermeulen, K., Van Bockstaele, D. R., and Berneman, Z. N. (2003). The cell cycle: a

review of regulation, deregulation and therapeutic targets in cancer. Cell

Proliferation 36, 131-149.

Vianello, F., and Dazzi, F. (2008). Mesenchymal stem cells for graft-versus-host

disease: a double edged sword? Leukemia 22, 463-465.

Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S. (2008). Functions

of natural killer cells. Nature Immunology 9, 503-510.

Voss, O. H., Kim, S., Wewers, M. D., and Doseff, A. I. (2005). Regulation of

monocyte apoptosis by the protein kinase Cδ-dependent phosphorylation of

caspase-3. Journal of Biological Chemistry 280, 17371-17379.

© CO

PYRI

GHT U

PM

123

Wahl, L. M., and Smith, P. D. (1991). Isolation of monocyte/macrophage populations.

Current Protocols in Immunology.

Wakitani, S., Imoto, K., Yamamoto, T., Saito, M., Murata, N., and Yoneda, M. (2002).

Human autologous culture expanded bone marrow mesenchymal cell

transplantation for repair of cartilage defects in osteoarthritic knees.

Osteoarthritis and Cartilage 10, 199-206.

Walker, F., and Burgess, A. W. (1985). Specific binding of radioiodinated

granulocyte-macrophage colony-stimulating factor to hemopoietic cells. The

EMBO Journal 4, 933.

Wang, Y., Chen, A.-D., Lei, Y.-M., Shan, G.-Q., Zhang, L.-Y., Lu, X., and Chen, Z.-

L. (2013). Mannose-binding lectin inhibits monocyte proliferation through

transforming growth factor-β1 and p38 signaling pathways. Plos One 8, 9

Wang, S., Qu, X., and Zhao, R. C. (2012). Clinical applications of mesenchymal stem

cells. Journal Hematol Oncology 5, 19.

Wang, T., and Brown, M. J. (1999). mRNA quantification by real time TaqMan

polymerase chain reaction: validation and comparison with RNase protection.

Analytical Biochemistry 269, 198-201.

Warnke, P., Springer, I., Wiltfang, J., Acil, Y., Eufinger, H., Wehmöller, M., Russo,

P., Bolte, H., Sherry, E., and Behrens, E. (2004). Growth and transplantation

of a custom vascularised bone graft in a man. The Lancet 364, 766-770.

Wilson, H. M. (2010). Macrophages heterogeneity in atherosclerosis–implications for

therapy. Journal of Cellular and Molecular Medicine 14, 2055-2065.

Wong, M. L., and Medrano, J. F. (2005). Real-time PCR for mRNA quantitation.

Biotechniques 39, 75.

Wüthrich, M., Ersland, K., Sullivan, T., Galles, K., and Klein, B. S. (2012). Fungi

subvert vaccine T cell priming at the respiratory mucosa by preventing

chemokine-induced influx of inflammatory monocytes. Immunity 36, 680-692.

WWW.miltenyibiotec.com

Xing, Z., Ohtoshi, T., Ralph, P., Gauldie, J., and Jordana, M. (1992). Human upper

airway structural cell-derived cytokines support human peripheral blood

monocyte survival: a potential mechanism for monocyte/macrophage

accumulation in the tissue. American Journal Respiration Cell Molecular

Biology 6, 212-218.

Yang, J., Zhang, L., Yu, C., Yang, X.-F., and Wang, H. (2014). Monocyte and

macrophage differentiation: circulation inflammatory monocyte as biomarker

for inflammatory diseases. Biomolecular Research 2, 1.

© CO

PYRI

GHT U

PM

124

Yang, M., Wei, X., Li, J., Heine, L. A., Rosenwasser, R., and Iacovitti, L. (2010).

Changes in host blood factors and brain glia accompanying the functional

recovery after systemic administration of bone marrow stem cells in ischemic

stroke rats. Cell Transplantation 19, 1073-1084.

Yusoff, Z, Maqbool, M., George, E., Hassan, R., Ramasamy, R (2016). Generation

and characterisation of human umbilical cord derived mesenchymal stem cells

by explant method. Medical Journal of Malaysia, 71(3) 105-110.

Zhang, B., Liu, R., Shi, D., Liu, X., Chen, Y., Dou, X., Zhu, X., Lu, C., Liang, W.,

and Liao, L. (2009). Mesenchymal stem cells induce mature dendritic cells into

a novel Jagged-2–dependent regulatory dendritic cell population. Blood 113,

46-57.

Zhang, Q. Z., Su, W. R., Shi, S. H., Wilder‐ Smith, P., Xiang, A. P., Wong, A., Nguyen, A. L., Kwon, C. W., and Le, A. D. (2010). Human gingiva‐ derived mesenchymal stem cells elicit polarization of M2 macrophages and enhance

cutaneous wound healing. Stem Cells 28, 1856-1868.

Zhang, W., Ge, W., Li, C., You, S., Liao, L., Han, Q., Deng, W., and Zhao, R. C.

(2004). Effects of mesenchymal stem cells on differentiation, maturation, and

function of human monocyte-derived dendritic cells. Stem Cells and

Development 13, 263-271.

Zhao, Q., Ren, H., and Han, Z. (2015). Mesenchymal stem cells: Immunomodulatory

capability and clinical potential in immune diseases. Journal of Cellular

Immunotherapy 2, 3-20.

Zhao, R. C. (2013). "Essentials of mesenchymal stem cell biology and its clinical

translation," Book Springer pages 3-15.

Zhou, L., Somasundaram, R., Nederhof, R. F., Dijkstra, G., Faber, K. N.,

Peppelenbosch, M. P., and Fuhler, G. M. (2012). Impact of human granulocyte

and monocyte isolation procedures on functional studies. Clinical and Vaccine

Immunology 19, 1065-1074.

Ziegler-Heitbrock, L., Ancuta, P., Crowe, S., Dalod, M., Grau, V., Hart, D. N.,

Leenen, P. J., Liu, Y.-J., MacPherson, G., and Randolph, G. J. (2010).

Nomenclature of monocytes and dendritic cells in blood. Blood 116, e74-e80.

Ziegler‐ Heitbroc, H., Thiel E., Futterer, A., Herzog, V., Wirtz, A., and Riethmüller, G. (1988). Establishment of a human cell line (Mono Mac 6) with

characteristics of mature monocytes. International Journal of Cancer 41, 456-

461.

© CO

PYRI

GHT U

PM

125

APPENDIX A

RNA extraction and quantification (RNeasy Plus Mini kit)

1) Cells harvested. 2) Buffer RLT Plus was added to cell pellet (350 µl for ‹5x106 cells and 600µl

for 5x106-1x107 cells).

3) Cells were homogenized by passing the lysate through 20-gauge needle (9.0 mm diameter) (5 times).

4) Homogenized lysate was transferred to eliminator spin column placed in a 2 ml collection tube and was centrifuged for 30 s at ≥ 8000×g (Flow through was

saved).

5) One volume (usually 350 µl or 600 µl) of 70% ethanol was added to the flow through, and mixed.

6) Up to 700 µl of the sample was transferred to the RNeasy spin column placed in a 2 ml collection tube and centrifuged for 15s at ≥ 8000×g (Flow through

was discarded).

7) Buffer RW 1 (700 µl) was added to the RNeasy spin column, and centrifuged for 15s at ≥ 8000×g (Flow through was discarded).

8) 500 µl buffer was added RPE to the RNeasy spin column, and was centrifuged for 15s at ≥ 8000×g (Flow through was discarded).

9) Buffer RPE (500 µl) was added to the RNeasy spin column, and centrifuged for 2 min at ≥ 8000×g.

10) RNeasy spin column was placed in new 2 ml collection tube and centrifuged for 1 min at ≥ 8000×g.

11) RNeasy spin column was placed in a new 1.5 ml collection tube, then 30 µl of RNase-free water was added to the column, and centrifuged for 1 min at ≥

8000×g.

© CO

PYRI

GHT U

PM

126

APPENDIX B

Complementary DNA (cDNA) synthetizing protocol

(Roche® cDNA synthesis kit, Germany)

1. 10ng - 5000ng of RNA were added to 2uL Random Hexamer Primer and toped up with PCR grade water (until 13uL).

2. The template-primer mixture was denatured by heating the tube in the heat block (at 65oC for 10 minute).

3. The tube was cooled on ice (for 5 minute). 4. Master Mix was add (7ul; Table 1). 5. Mixture was mixed well and centrifuged. 6. Sample was incubated (at 25oC for 10 minute followed by 55oC for 30 minute). 7. Transcriptor reverse transcriptase was inactivated via heating (at 85oC for 5

minute).

8. The reaction was stopped by cooling (on ice for 5 minutes). 9. The reaction tube was stored (at 2 - 8oC for 1-2 hours OR at -15oC to - 25oC

for longer period of time).

A1: Reverse Transcription Master Mix Composition

APPENDIX C

The protocol of RT-qPCR (Roche, SYBR green, Germany)

1. PCR reaction mixture was prepared (total volume 20µl) using 1.5ml reaction tube on ice (Table 2).

2. The PCR reaction mixture (15µl) was added per well in multi-well plate. 3. The cDNA template (5µl) was added. 4. Multi-well plate was sealed with multi-well sealing foil. 5. Multi-well plate was replaced into the plate holder of LightCycler® 480

Instrument.

6. PCR experimental program was started as described in Table 3.

Reagent Volume

5x Transcriptor reverse transcriptase reaction buffer 4uL

Protector RNase inhibitor 0.5uL

Deoxynucleotide mix 2uL

Transcriptor reverse transcriptase 0.5uL

Total volume for 1 reaction 7uL

© CO

PYRI

GHT U

PM

127

A2: PCR reaction mixture

Reagent Volume

PCR primer (Forward+ Reverse) 2µL

SYBRgreen Master Mix 10µL

cDNA template 1µL

PCR grade water 7µL

Total 20µL

A3: PCR Primer Sequence for RT-qPCR

A4: PCR experimental program

Program Cycles Temperature target (oC)

Pre-incubation 1 95

Amplification

Annealing

95

56

Melting curve 1 65-98

Cooling 1 40

Gene Name 5’-3’ Sequence Annealing

Temperature

TNFRSF11A Forward 5’-TCTACTCTCTTTCCAAGGAAGGT-3’ 540C

Reverse

5’-CAGCTCAACAAGGACACAGT-3’

TGFA Forward 5’-TGATGGCCTGCTTCTTCTG-3’ 540C

Reverse

5’-ACACTCAGTTCTGCTTCCAT-3’

FGFR1 Forward 5’-ACACCTTACACATGAACTCCAC-3’ 600C

Reverse

5’-AGCATCAACCACACATACCAG-3’

C3 Forward 5’-AGTCTCCTGCTTTAGTGATGC-3’ 600C

Reverse 5’-GCCTTTGTTCTCATCTCGCT-3’

45

45

© CO

PYRI

GHT U

PM

128

APPENDIX D

A1: Immunophenotyping of THP-1 and U937 using various myeloid cell surface markers

Flow cytometric representation of monocyte immunophenotyping using panel of antibodies conjugated with fluorochrome and analysed using BD

FACS Fortessa flow cytometer and 1 x 104 cells were acquired and analysed using BD FACS Diva Software.

© CO

PYRI

GHT U

PM

129

APPENDIX E

A2: Immunophenotyping of THP-1 differentiation using various myeloid

cell surface markers

One million (106) cells were analysed for differentiation. (A) unstain THP-1 served as

control. (B) Immunophenotyping of THP-1 differentiation into DC and MAC in the

presence and absence of MSC. Immunophenotyping was performed with a panel of

antibodies conjugated with a fluorochrome, using BD FACS Fortessa flow cytometer

and 1 x 104 cells were acquired and analysed using BD FACS Diva Software.

© CO

PYRI

GHT U

PM

130

BIODATA OF STUDENT

Maryam Maqbool was born in Pakistan on the 3rd of December 1986. After completing

her upper primary and secondary education in the Australian International School in

Malaysia with honours, she pursued a degree in Biotechnology at University Putra

Malaysia. Upon completion of her bachelor degree, she was offered a place to continue

her master in Immunobiology from the Faculty of Medicine and Health Sciences

University Putra Malaysia, under the supervision of Dr Rajesh Ramasamy. During her

tenure as a master’s student, she received several awards for her work, at the Research

Competition (PRPI) organized by the University. She then worked towards a PhD in

Immunology. Maryam Maqbool is also actively involved in University Putra Malaysia

International Student Association (UPMISA); and is a student member of the Tissue

Engineering and Regenerative Society of Malaysia (TESMA).

© CO

PYRI

GHT U

PM

131

LIST OF PUBLICATIONS

R Ramasamy, K Krishna, M Maqbool, S. Vellasamy, VH Sarmadi, M Abdullah & S

Vidyadaran. (2010). The Effect of Human Mesenchymal Stem Cell on

Neutrophil Oxidative Burst. Malaysian Journal of Medicine and Health

Sciences 6:11-17

Rajesh Ramasamy, M.Maqbool, Abdul Latiff Mohamed & Rahim MD. Noah. (2010).

Elevated neutrophil respiratory burst activity in essential hypertensive

patients. Cellular biology 263:230-234

M.Maqbool, E.George, S.Vidyadaran & R.Ramasamy. (2011). Human Mesenchymal

Stem Cells Protect Neutrophils from Serum Deprived Cell Death. Cell

Biology International 35, 1247-1251

M.Maqbool, S.Vidyadaran, E.George & R.Ramasamy. (2011). Optimisation of

Laboratory Procedures for Isolating Human Peripheral Blood Derived

Neutrophils. Malaysian Journal of Medicine and Health Sciences 66, 296-

299

Mohadese Hashem Borojer