universiti putra malaysia isolation and …psasir.upm.edu.my/4867/1/fbsb_2007_6a.pdfvarious types of...

UNIVERSITI PUTRA MALAYSIA

ISOLATION AND CHARACTERIZATION OF MESENCHYMAL CELLS FROM HUMAN BONE MARROW AND THEIR DEVELOPMENT INTO

BONE CELLS

WAN NAZATUL SHIMA BT SHAHIDAN

FBSB 2007 6

1

ISOLATION AND CHARACTERIZATION OF MESENCHYMAL CELLS FROM

HUMAN BONE MARROW AND THEIR DEVELOPMENT INTO BONE CELLS

By

WAN NAZATUL SHIMA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

April 2007

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Dedication

Special thanks are dedicated to my loving parents, Shahidan bin Mohd and Sharipah

Norida bt Syed Nordin for their moral support and prayers. Not forgetting to my dearest

husband, Ahmad Amharie bin Zainal Bakri and our daughter Nur Alya Humaira for their

understanding, support and enriching love during my endeavour. I love you all.

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

requirement for the degree of Master of Science

ISOLATION AND CHARACTERIZATION OF MESENCHYMAL CELLS FROM

HUMAN BONE MARROW AND THEIR DEVELOPMENT INTO BONE CELLS.

By


April 2007

Chairman: Professor Abd Manaf Ali, PhD

Faculty: Biotechnology and Biomolecular Sciences

Everyday thousands of people suffer from bone diseases that lead to destruction of bone

tissue. Various types of bone allograft, xenograft and synthetic biomaterial are now used

for bone replacement therapy. The future of bone reconstruction lies in the use of stem

cell technology for bone development. Within the bone marrow stroma there are exists a

subset of nonhematopoietic cells referred to as mesenchymal stem cells (MSCs) or

mesenchymal progenitor cells (MPCs). These cells can be expanded ex vivo and induced,

either in vitro or in vivo, to terminally differentiate into osteoblasts, chondrocytes,

adypocytes, tenocytes, myotubes, neural cells, and hematopoietic supporting stroma. The

multipotential of these cells, their easy collection and culture, as well as their high ex vivo

expansive potential makes these cells an attractive therapeutic tool. The purpose of this

study was to isolate, expand and characterize mesenchymal stem cell from human bone

marrow in Mesenchymal Stem Cell Growth Medium (MSCGM), to compare the cell

growth in the MSCGM and Dulbecco Modified Eagle‟s Medium (DMEM) with 10%

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Fetal Bovine Serum (FBS) medium, to differentiate MSCs into osteoblast cells and to

determine the osteogenic potential of the differentiated MSC by detecting the expression

of osteoblast-specific genes such as collagen type 1, osteocalcin, runx 2, osteopontin and

alkaline phosphatase. In this study, MSCs were isolated from human bone marrow and

cultured in MSCGM medium and DMEM-10% FBS medium. Culture-expansion of

MSCs was characterized by the presence of CD 105 marker using Labelled Streptavidin

Biotin (LSAB) method. The MSCs were cultured in the MSCGM and DMEM-10% FBS

medium within five days. The MSCs were then cultured in osteogenic medium

containing DMEM medium supplemented with Fetal Bovine Serum (FBS), antibiotics,

ascorbic acid, β-glycerol phosphate and dexamethasone to differentiate into osteoblasts.

Their osteogenic differentiation was determined with the formation of a mineralized

extracellular matrix visualized by Von Kossa staining and Alkaline Phosphatase (ALP)

assay. Moreover, osteogenic differentiation was also judge by RT-PCR profiling of

osteoblast gene expression. MSC first attached to the dish surface and exhibited

fibroblast-like spindle shape. The cells were also identified as MSCs based on their

immunophenotype of CD 105 which resulted in funchia coloured staining. The MSCs

culture in MSCGM medium expanded and proliferated rapidly compared to MSCs

culture in DMEM-10% FBS medium. Incubation of bone marrow-derived MSCs in the

osteogenic medium for 3 weeks resulted in a dramatic increase in ALP activity and

accumulation of calcium deposit, as assessed by ALP assay and Von Kossa staining,

respectively. This osteogenic potential upon culture in osteogenic medium was further

confirmed by the RT-PCR analysis where the expressions of osteoblast specific genes

were confirmed by molecular weight produced on agarose gel. We conclude that

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MSCGM is the best choice for expanding and proliferating MSCs and suggest that MSC

from bone marrow have pure osteogenic potential and have the capability to differentiate

into osteoblast. This potential assures that bone marrow can be a legitimate source of

MSCs for production of osteoblast which can be utilized in bone replacement therapy.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PEMENCILAN DAN PENCIRIAN SEL MESENKIMA DARIPADA TULANG

SUM-SUM MANUSIA DAN PERKEMBANGANNYA KEPADA SEL-SEL

TULANG.

Oleh


April 2007

Pengerusi: Profesor Abd Manaf Ali, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Setiap hari hampir beribu manusia mati menderita penyakit tulang disebabkan oleh

kerosakan tisu tulang. Pelbagai jenis allograf, xenograf tulang dan tulang sintetik telah

digunakan untuk terapi penggantian tulang. Pembinaan semula tulang bergantung kepada

teknologi sel stem. Di dalam tulang sum-sum terdapat subset sel bukan hematopoitik

yang dirujuk sebagai sel mesenkima atau generasi sel mesenkima. Sel-sel ini boleh

dikembangbiakkan atau diaruhkan samada in vitro atau in vivo dan akhirnya bertukar

kepada osteoblas, chondrosit, adiposit, tenosit, myotiub, sel neuro dan tisu penyokong

hematopoitik. Oleh kerana sel ini mempunyai pelbagai potensi, mudah dikumpulkan dan

dikultur, juga mempunyai daya kembang biak ex vivo yang tinggi, menjadikan sel ini

sebagai satu alat terapeutik yang menarik. Tujuan kajian ini adalah untuk memencilkan,

mengembang biakkan dan mencirikan sel mesenkima daripada tulang sum-sum di dalam

media MSCGM, membandingkan perkembangan sel di dalam media MSCGM dan media

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DMEM-10% FBS, menukarkan sel mesenkima kepada sel osteoblas dan menentukan

kebolehan osteogenik sel mesenkima bertukar kepada sel osteoblas melalui ekspresi lima

gen khusus osteoblas; collagen type 1, osteocalcin, runx 2, osteopontin dan alkaline

phosphatase. Di dalam kajian ini sel stem mesenkima telah dipencilkan dan dikultur di

dalam media MSCGM dan media DMEM-10% FBS. MSC kultur yang berkembang biak

telah diuji sifat dan ciri-cirinya melalui kehadiran penanda permukaan sel CD 105 dengan

kaedah Labelled Streptavidin Biotin (LSAB). Sel mesenkima telah dikultur di dalam

media MSCGM dan juga media DMEM-10% FBS dalam lingkungan lima hari. Sel

mesenkima kemudiannya dikulturkan di dalam media DMEM dengan tambahan Fetal

Bovine Serum (FBS), antibiotic, askorbik asid, β-glycerol Phosphate dan dexamethasone

untuk membezakan kepada sel osteoblas. Pembezaan osteogenik yang optimum telah

ditentukan daripada pembentukan matrik mineral ekstraselular yang dilihat secara visual

melalui tompokan Von Kossa dan pegujian Alkaline Phosphatase (ALP). Tambahan lagi

pembezaan osteogenik telah diadili melalui ekspresi osteoblas gen profil RT-PCR. Sel

mesenkima pada mulanya melekat pada permukaan bekas kultur dan menunjukkan

bentuk seperti gelungan fibroblast. Sel ini juga telah dikenal pasti sebagai MSC

berdasarkan pada immunofenotip (CD105), menyebabkan pewarnaan warna funchia. Sel

mesenkima yang dikultur di dalam media MSCGM tumbuh dah berkembang biak dengan

cepat berbanding dengan sel mesenkima yang dikultur di dalam media DMEM-10% FBS.

Pengeraman MSC daripada sel tulang sum-sum di dalam media osteogenik selama tiga

minggu menghasilkan peningkatan dalam aktiviti ALP dan pengumpulan mendapan

kalsium melalui penilaian pengujian ALP dan tompokan Von Kossa. Potensi osteogenik

telah disahkan lagi menggunakan analisis RT-PCR di mana ekspresi gen khusus telah

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menunjukkan jaluran DNA di atas gel agarose. Kami merumuskan bahawa media

MSCGM adalah pilihan terbaik untuk pertumbuhan dan kembang biak sel mesenkima

dan mencadangkan bahawa sel mesenkima daripada tulang sum-sum mempunyai potensi

osteogenik yang tulen dan berkeupayaan untuk bertukar kepada sel osteoblas. Potensi

mesengenic ini meyakinkan bahawa sel stem daripada tulang sum-sum adalah

multipotensi MSCs dan menunjukkan bahawa tulang sum-sum merupakan sumber MSC

yang sah untuk menghasilkan osteoblas yang dapat digunakan dalam terapi penggantian

tulang.

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ACKNOWLEDGEMENTS

My deepest appreciation is dedicated to my main supervisor Professor Abd. Manaf Ali

for his advice and support throughout my study, my co-supervisor Dr. Noorjahan Banu

Alitheen and Dr. Muhajir Hamid as well. I am also greatly indebted to Professor Dr. Abd.

Rani Samsudin, Dean of Dental School at Universiti Sains Malaysia Kubang Kerian

Kelantan for his continuous assistance and guidance.

I would like to express my utmost gratitude to USM‟s Craniofacial Biology Laboratory

staff, Human Genome Center staffs especially science officer Cik Azlina Ahmad and all

post graduate student, also staff and all post graduate student of Biology Cell Laboratory,

UPM for their hospitality and help whenever is needed.

Not forgetting, this acknowledgement goes to Operation Theater Unit staffs in HUSM

who gave a great help in obtaining human bone marrow samples.

Last but not least, special thanks to Universiti Sains Malaysia for Academic Staffs

Teaching Scholarships (ASTS) for my study.

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I certify that an Examination Committee has met on 2nd

April to conduct the final

examination of Wan Nazatul Shima bt Shahidan on her Master of Science thesis entitled

“Isolation and Characterization of Mesenchymal Stem Cell from Bone Marrow and Their

Development Into Bone Cells.” in accordance with Universiti Pertanian Malaysia (Higher

Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981.

The Committee recommends that the candidate be awarded the relevant degree. Members

of the Examination Committee are as follows:

Dr. Nor Aripin Shamaan

Professor

Biochemistry Department

Fakulty of Bioteknology dan Biomolecule Sciences

Universiti Putra Malaysia

(Chairman)

Dr. Rozita Rosli

Associate Professor

Fakulti Perubatan dan Sains Kesihatan


(Internal Examiner)

Dr. Md. Zuki Abu Bakar

Associate Professor

Praclinical Veterinary Science Department

Fakultiy of Medical Veterinar


(Internal Examiner)

Dr. Ruszymah Bt. Hj. Idrus

Professor

Fisiology Department

Universiti Kebangsaan Malaysia

(External Examiner)

_________________________________

HASANAH MOHD. GHAZALI, PhD

Professor/Deputy Dean

School of Graduate Studies


Date: 21 JUNE 2007

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

fulfilment of the requirement for the degree of Master of Science. The members of the

Supervisory Committee are as follows:

Abd. Manaf bin Ali, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences


(Chairman)

Noorjahan Banu Alitheen, PhD

Lecturer



(Member)

Muhajir Hamid, PhD

Lecturer



(Member)

Abd. Rani Samsudin, PhD

Lecturer

Pusat Pengajian Sains Pergigian

Universiti Sains Malaysia.

(Member)

____________________

AINI IDERIS, PhD

Professor/ Dean

School of Graduate Studies


Date: 17 JULY 2007

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DECLARATION

I hereby declare that the thesis is based on my original work except quotations and

citations, which have been duly acknowledged. I also declare that it has not been

previously or concurrently submitted for any other degree at UPM or other institutions.

____________________________________


Date: 29 MAY 2007

13

TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xix

CHAPTER

1

INTRODUCTION 21

2 LITERATURE REVIEW 26

2.1 Stem Cells 26

2.2 Bone Marrow 27

2.3 Bone Cells 28

2.4 Mesenchymal Stem Cells 30

2.4.1 Sources of Mesenchymal Stem Cells (MSCs) 31

2.4.2 Existence of Mesenchymal Stem Cells (MSCs) 34

2.4.3 MSC Differentiation to the Osteoblast Lineage 35

2.4.4 Markers of Osteoblast 36

2.4.5 Gene and Protein Expression by MSCs 37

2.5 Cell Biology of Bone Growth 40

2.5.1 The Structure and Function of Bone 40

2.5.2 Bone and Mesenchymal Stem Cells 41

2.5.3 Skeletal Morphogenesis and Growth

41

3 MATERIALS AND METHODS 44

3.1 Isolation and Culture of Mesenchymal Stem Cell 44

3.2 Immunohistostaining of Cells with CD 105 Using Labelled

Streptavidin Biotin (LSAB)

45

3.3 Mesenchymal Stem Cell Culture in MSCGM and DMEM-

10% FBS

46

3.4 Osteogenic differentiation 47

3.5 Matrix mineralization 47

3.5.1 Von Kossa Staining

3.5.2 Alkaline Phosphatase Assay

47

48

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3.6 Analysis Gene Expression of Osteoblast- Derive

Mesenchymal Stem Cell

49

3.6.1 Sample Preparation 49

3.6.2 RNA Extraction Procedure Using a Commercial Kit 49

3.6.3 Quantification of RNA 51

3.6.4 Reverse Transcriptase and Polymerase Chain

Reaction (RT-PCR) Using Commercial Kit

52

4 RESULTS 56

4.1 Culture of Human Bone Marrow Mesenchymal Stem Cells 56

4.2 Characterization of Mesenchymal Stem Cell

Using LSAB Kit

58

4.3 Human mesenchymal stem cell in MSCGM and DMEM-

10% FBS medium.

60

4.4 Human mesenchymal stem cells in osteogenic medium. 61

4.5 Differentiation of MSC 63

4.5.1 Von Kossa Staining 63

4.5.2 Alkaline Phosphatase Assay

4.5.3 Gene Expression

4.5.3.1 RNA Quantification

65

66

66

5 DISCUSSION 70

5.1 Isolation of MSCs from Human Bone Marrow 70

5.2 In Vitro Differentiation of Human Bone

Marrow MSCs

71

5.3 RNA Extraction 74

5.4 Osteoblast Gene Expression

76

6 GENERAL DISCUSSION AND CONCLUSION 79

6.1 General Discussion 79

6.2 Conclusion

80

REFERENCES 82

APPENDICES 92

BIODATA OF THE AUTHOR 102

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LIST OF TABLES

Table

Page

1 Serial dilution of p-nitrophenol standard 200 μg/ml for

concentration from (A) 0 μg/ml to (H) 200 μg/ml

49

2 Volume of RNA Lysis Solution for each sample 51

3 Volume of RNase-free water for each sample 51

4 Reaction components for one-step RT-PCR 54

5 Thermal cycling conditions

54

6 RT-PCR primers for validation of gene expression 55

7 RNA concentration of differentiated MSCs in osteogenic

medium and purity of the nucleic acid in day 0, day 2, day

5, day 7, day 14, day 21 and day 28.

66

16

LIST OF FIGURES

Figure Page

1 The differentiation pathways of MSCs 15

36

2 Morphology of mesenchymal stem cells. (A) Early

morphology of mesenchymal stem cell after overnight cultured

at x100 magnification. (B) x100 magnification of

Mesenchymal stem cell after 5 days cultured. (C) x200

magnification of confluent mesenchymal stem cells. (D) The

appearance and growth of mesenchymal stem cell in DMEM-

10% medium at x200 magnification

57

3 Colored stained of mesenchymal stem cells (A) x200

magnification of human bone marrow mesenchymal stem cell

with CD 105 primary antibody resulting in an insoluble

fuchsin-colored precipitate at the antigen site (B) x200

magnification of human bone marrow mesenchymal stem cell

line without CD 105 primary antibody resulting no insoluble

fuchsin-colored precipitate at the antigen site (negative

control). (C) x200 magnification of human bone marrow

mesenchymal stem cell line with CD 105 primary antibody

resulting in an insoluble fuchsin-colored precipitate at the

antigen site (positive control)

59

4 The effect of two different media on mesenchymal stem cells

growth. Growth curves were established by daily counts for 5

days. This data represent the mean of independent experiment

within each individual sample was counted in triplicate.

60

5

Effect of osteogenic medium on cell morphology. (A) x100

magnified mesenchymal stem cells day 0 in MSCGM

demonstrates the spindle-shape morphology. (B) x100

magnified mesenchymal stem cells day 2 in osteogenic

medium proliferate and remains the spindle-shape

morphology. (C) and (D) Cell grown with osteogenic medium

have become polygonal and more numerous on day 5 and day

7 respectively at x200 magnification. (E), (F) and (G) x200

magnified mesenchymal stem cells day 14, day 21 and day 28

respectively exhibit the cells combine each other and began to

form nodular aggregates in osteogenic medium.

61

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6 MSC-driven osteogenesis in monolayer cultures. (A) Positive

Von Kossa staining in monolayer culture of human BM-

derived MSCs exposed to osteogenic medium for 3 weeks

shown at x400 magnification. (B) Control monolayer cultures

of MSCs grown with basic medium (no osteogenic

supplement) for 3 weeks showed negative reaction to Von

Kossa staining at x400 magnification

64

7 Alkaline phosphatase activity of human mesenchymal stem

cell. Results are based on three individual experiments. The

data are the means of three independent experiments within

which each individual sample was analyze in triplicate.

65

8 Agarose gel electrophoresis of RT-PCR product from

osteoblast derived mesenchymal stem cell cultures using

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

forward and reverses primers as the positive control. Lane M;

100 bp DNA ladder (Promega, Maddison, USA); Lane 1, Day

0; Lane 2, Day 7; Lane 3, Day 14; Lane 4, Day 21; Lane 5,

Day 28. The size of amplified products is 731 bp.

67



Collagen Type 1 forward and reverse primers. Lane M; 100 bp

DNA ladder (Promega, Maddison, USA); Lane 1, Day 0; Lane

2, Day 7; Lane 3, Day 14; Lane 4, Day 21; Lane 5, Day 28.

The size of amplified products is 306 bp.

67



Osteocalcin forward and reverse primers. Lane M; 100 bp




68

18


osteoblast derived mesenchymal stem cell cultures using bone

sialoprotein (BSP), runt-related transcription factor-2 (Runx

2/Cbfa1) forward and reverse primers. Lane M; 100 bp DNA

ladder (Promega, Maddison, USA); Lane 1, Day 0; Lane 2,

Day 7; Lane 3, Day 14; Lane 4, Day 21; Lane 5, Day 28. The

size of amplified products is 261 bp

68



Osteopontin forward and reverse primers. Lane M; 100 bp




68



Alkaline Phosphatase forward and reverse primers. Lane M;

100 bp DNA ladder (Promega, Maddison, USA); Lane 1, Day

0; Lane 2, Day 2; Lane 3, Day 5; Lane 4, Day 7; Lane 5, Day

14. The size of amplified products is 476 bp.

69

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LIST OF ABBREVIATIONS

ALP Alkaline Phosphatase

BMC Bone marrow derived cell

BM-MSC Bone Marrow Mesenchymal Stem Cell

CPD Citrate Phosphate Buffer

DMEM Dulbecco‟s Modified Eagle Medium

dNTP deoxynucleotide triphosphate

ECM Extracellular matrix

FBS Fetal Bovine Serum

GPDH Glycerol-3-Phosphate-Dehydrogenase

HLA Human Leucocyte Antigen

HSCs Haematopoietic Stem Cells

IFNγ Interferon Gamma

LSAB Labelled Streptavidin Biotin

MDA Muscular Dystrophy Association

MNC Mononuclear Cell

MSCGM Mesenchymal Stem Cell Growth Medium

MSCs Mesenchymal Stem Cells

PBS Phosphate Buffered Saline

PBSCs Peripheral Blood Stem Cells

PCR Polymerase Chain Reaction

RNA Ribonucleic Acid

20

RT-PCR Reverse Transcriptase Polymerase Chain Reaction

Runx 2 Runt-related transcription factor-2

UCB Umbilical Cord Blood

β-ME β-mercaptoethanol

21

CHAPTER 1

INTRODUCTION

A stem cell is a cell that has the ability to divide (self-replicate) for indefinite periods

often throughout the life of the organism. Under the right conditions, or given the right

signals, stem cells can give rise (differentiate) to the many different cell types that make

up the organism. Stem cells have the potential to develop into mature cells that have

characteristic shapes and specialized functions, such as bone cells, heart cells, skin cells

or nerve cells (Chandross et al., 2001).

Many of the terms used to define stem cells depend on the behavior of the cells in the

intact organism (in vivo), under specific laboratory conditions (in vitro), or after

transplantation in vivo, often to a tissue that is different from the one from which the stem

cells were derived. For example, the fertilized egg is said to be totipotent from the latin

word totus, meaning entire because it has the potential to generate all the cells and tissues

that make up an embryo and that support its development in utero. The fertilized egg

divides and differentiates until it produces a mature organism. The term pluripotent used

to describe stem cells that can give rise to cells derived from all three embryonic germ

layers; mesoderm, endoderm, and ectoderm. These three germ layers are the embryonic

source of all cells of the body. All of the many different kinds of specialized cells that

make up the body are derived from one of these germ layers. “Pluri” derived from the

latin word plures that means several or many. Thus, pluripotent cells have the potential to

22

give rise to several type of cell, a property observed in the natural course of embryonic

development and under certain laboratory conditions. Unipotent stem cell, a term usually

applied to a cell in adult organisms, means that the cells in question are capable of

differentiating along only one lineage. “Uni” is derived from the latin word unus, which

means one. Also, it may be that the adult stem cells in many differentiated, undamaged

tissues are typically unipotent and give rise to just one cell type under normal conditions.

This process would allow for a steady state of self-renewal for the tissue. However, if the

tissue becomes damaged and the replacement of multiple cell types is required,

pluripotent stem cells may become activated to repair the damage.

Embryonic stem cell and adult stem cell are two types of stem cell. The embryonic stem

cell is defined by its origin that is from one of the earliest stages of the development of

the embryo, called blastocyst. Specifically, embryonic stem cells are derived from the

inner cell mass of the blastocyst at a stage before it would implant in the uterine wall. It

can give rise to cells derived from all three germ layers. The adult stem cell is an

undifferentiated (unspecialized) cell that is found in a differentiated (specialized) tissue;

it can renew itself and become specialized to yield all of the specialized cell types of the

tissue from which it originated. Adult stem cells have been found in the bone marrow,

blood stream, cornea and retina of the eye, the dental pulp of the tooth, liver, skin,

gastrointestinal tract, and pancreas (Slack et al., 2000). Unlike embryonic stem cells, at

this point in time, there are no isolated adult stem cells that are capable of forming all

cells of the body.

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Adult stem cell compartment consists of two sub-populations which are hematopoietic

stem cells (HMCs) and mesenchymal stem cells (MSCs). A hematopoietic stem cell is a

cell that form blood and immune cells. They are responsible for the constant renewal of

blood and the production of new blood cells each day. The cells are isolated from the

blood or bone marrow that can renew itself, can differentiate to a variety of specialized

cells, can mobilize out of the bone marrow into circulating blood, and can undergo

programmed cell death, called apoptosis; a process by which cells that are detrimental or

unneeded undergo self-destruct. Adult mesenchymal stem cells are adult human

pluripotent progenitor cells found in bone marrow, peripheral blood, cord blood, adipose

tissue and liver. They have self-renewal capacity without differentiation in long term

culture. Under certain conditions, MSC could be differentiated into adipocytes,

chondrocytes, astrocytes, tenocytes, cardiomyocytes, hepatocytes, neurons, muscle cells,

endothelial and endodermal cells (Sanchez-Ramos et al., 2000). MSCs have generated a

great deal of excitement and promise as a potential source of cells for cell-based

therapeutic strategies, primarily owing to their intrinsic ability to self-renew and

differentiate into functional cell types that constitute the tissue in which they exist. MSCs

are considered a readily accepted source of stem cells because such cells have already

demonstrated efficacy in multiple types of cellular therapeutic strategies, including

applications in treating children with osteogenesis imperfecta (Horwitz et al., 2002),

haematopoietic recovery (Koc et al., 2000), and bone tissue regeneration strategies (Petite

et al., 2000).

24

Despite the great interest in MSC, there is still no established protocol for isolation and

expansion of the cells in culture. In most experiments isolated MSC from bone marrow

mononuclear cells were determined based on their tight adherence to tissue culture

plastic. The isolated cells were initially heterogenous, and were difficult to be

distinguished from other adherent cells. Several methods have been developed to prepare

more homogenous populations but none of protocols have earned wide acceptance (Hung

et al., 2002). However, today there are many studies have isolated MSCs and controlled,

in vitro, its differentiation into cartilaginous tissue and bone using new technology for

repairing injured tissues of mesenchymal origin (Renata et al., 2006)

In this study, we have tried a method according to Friedenstein (1982) who was the first

to identify a fibroblast-like cell in the bone marrow that can be cultured undifferentiated

in vitro (Bruder et al., 1994) with modification in terms of media use and have

succesfully differentiated it into bone cells. Bone formation in the embryo, and during

adult fracture repair and remodeling, involves the progeny of cells called mesenchymal

stem cells. These cells continuously replicate themselves, while a portion become

commited to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and

muscle. The differentiation of these cells, within each lineage, is a complex multistep

pathway involving discrete cellular transitions much like that, which occurs during

hematopoiesis. Progression from one stage to the next depends on the presence of

specific bioactive factors, nutrient and other environmental cues whose exquisitely

controlled contributions orchestrate the entire differentiation phenomenon (Price et al.,

1993).

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