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
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
2
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.
3
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
WAN NAZATUL SHIMA BT SHAHIDAN
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
WAN NAZATUL SHIMA BT SHAHIDAN
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.
10
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
Universiti Putra Malaysia
(Internal Examiner)
Dr. Md. Zuki Abu Bakar
Associate Professor
Praclinical Veterinary Science Department
Fakultiy of Medical Veterinar
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Universiti Putra Malaysia
(Chairman)
Noorjahan Banu Alitheen, PhD
Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Muhajir Hamid, PhD
Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Abd. Rani Samsudin, PhD
Lecturer
Pusat Pengajian Sains Pergigian
Universiti Sains Malaysia.
(Member)
____________________
AINI IDERIS, PhD
Professor/ Dean
School of Graduate Studies
Universiti Putra Malaysia
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.
____________________________________
WAN NAZATUL SHIMA BT SHAHIDAN
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
14
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
15
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
9 Agarose gel electrophoresis of RT-PCR product from
osteoblast derived mesenchymal stem cell cultures using
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
10 Agarose gel electrophoresis of RT-PCR product from
osteoblast derived mesenchymal stem cell cultures using
Osteocalcin 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 294 bp.
68
18
11 Agarose gel electrophoresis of RT-PCR product from
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
12 Agarose gel electrophoresis of RT-PCR product from
osteoblast derived mesenchymal stem cell cultures using
Osteopontin 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 702 bp.
68
13 Agarose gel electrophoresis of RT-PCR product from
osteoblast derived mesenchymal stem cell cultures using
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.
23
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).