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UNIVERSITI PUTRA MALAYSIA
PARVIN SALEHINEJAD
FBSB 2012 16
IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM
HUMAN UMBILICAL CORD
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IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM
HUMAN UMBILICAL CORD
By
PARVIN SALEHINEJAD
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy
June 2012
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Abstract of Thesis Presented to the Senate of Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy
IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM
HUMAN UMBILICAL CORD
By
PARVIN SALEHINEJAD
June 2012
Chairman: Noorjahan Banu Binti Mohamed Alitheen, PhD
Faculty: Biotechnology and Biomolecular Sciences
The Wharton’s Jelly (WJ) of the umbilical cord is a rich source of mesenchymal
stem cells (MSCs) with a various potential therapeutic applications. Human
umbilical cord mesenchymal stem cells (hUCMCs) can be isolated with different
methods, expanded and differentiated into neuron, astrocyte and oligodendrocyte in
vitro.
In the first study, four methods to isolate hUCMCs were compared. Three of them
were based on using an enzyme cocktail containing collagenase, hyaluronidase and
trypsin (CHT), collagenase /trypsin (CT) or trypsin alone (TRP) and the other was
based on the explant culture (Exp) method. In the second experiment, the
competence of DMEM/ F12 and alpha-MEM/Glutamax (α-MEM/GL), also the
epidermal growth factor (EGF) and fibroblast growth factor (FGF) role to expand
hUCMCs were evaluated. Finally, hUCMCs were examined for their differentiation
rate into neural lineage cells by six various cocktails made of a base media
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(DMEM/LG), 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), EGF and
FGF.
Comparison of the four isolation methods of the hUCMCs showed that more cells
were isolated in the CT, CHT and TRP groups, respectively. Cells were successfully
isolated in TRP group but they did not propagate normally. Flow cytometry analysis
revealed that CD44, CD73, CD90 and CD105 were expressed on the cell surface in
all groups, but there was no expression of hematopoietic lineage markers, CD34 and
CD45, in any of the groups. In addition, the expression of C-kit and OCT-4 in
enzymatic (CT and CHT) groups was greater than Exp group. One-way ANOVA
showed that cell activity rate in the Exp group was significantly higher (P<0.001)
than the other groups. A positive alkaline phosphatase activity and differentiation to
adipogenic and osteogenic lineage was detected in the hUCMCs of all groups.
The ratio of CD44 and CD105 expression cells were similar in DMEM/F12 and α-
MEM/GL groups, while in both of the groups expression of the hematopoietic cells
surface marker CD34 were negative. Differentiation potential into adipogenic and
osteogenic cells were similar in both groups. Student`s t-test for evaluation of cell
metabolism with WST-1 showed that the quality of the expanded cells in α-
MEM/GL group was significantly higher (P<0.001) than DMEM/F12
group. Population doubling time (PDT) was calculated at 60 hours for DMEM/F12
group, while it was 47 hours in α-MEM/GL group that this difference was
statistically significant (P<0.001). Data sets were compared using two- way
ANOVA.
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Semi-quantitative RT-PCR revealed that the TERT in hUCMs/EGF was 0.26 ± 0.06.
Cell cycle analysis showed that EGF has caused 37.34± 9.8% and 7.71± 0.53% of the
hUCMs to move in the S and G2/M phases, respectively. While, after exposure to
FGF 29.92± 2.55% and 9.92± 0.24% of the cells progressed to S and G2/M phases,
respectively.
Induction of neuronal differentiation with various cocktails showed that there was no
statistically significant difference (one-way ANOVA with a post hoc Tukey’s test)
among the groups. Despite, the cocktail consist of DMEM/LG, FBS, RA, FGF, and
EGF (DF/R/Fg/E group) resulted in the expression of the highest percentage of
nestin, ß-tubulin III, neurofilament, and CNPase 69.7 ± 6%, 76 ±1.7%, 89 ±9.5%,
and 16.3 ±5.2%, respectively. Also, in this group and the DF/Ds/Fg/E group, the
highest cell proliferation was observed. On the other hand, DF/Ds/Fg/E group had
the highest percentage of GFAP expression (36 ±12.5%). While, the expression
level of NF, GFAP, and CNPase was at least in the DF group. The least percentage
of nestin and ß-tubulin III expression was observed in the DF/Ds group.
It may be concluded that isolation technique affects both the amount and the quality
of the isolated hUCMCs. When collagenase/trypsin enzymes are used for isolation of
hUCMCs, the waiting time for primary culture is shorter, the number of isolated cells
is higher and pluripotency properties of them were greater. The type of culture media
also affects the quality of the harvested cells. α-MEM/GL supports hUCMCs growth
more strongly than DMEM/F12. Furthermore, growth factors also affect the TERT
expression and cell cycle phases. In this case, EGF increases TERT expression and
the number of cells that progress towards S/G2/M phases. In addition, hUCMCs
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differentiate into various neuronal cell types successfully; the amount of different
neuronal cell types is influenced by inducing agents. FGF and EGF are important
inducers for differentiation of hUCMCs into neuron, astrocyte and oligodendrocyte.
RA can induce hUCMCs to differentiate into neuron and oligodendrocyte; while in
contribution to astrocyte differentiation, DMSO had a pivotal role.
From this study, it can be concluded that different isolation methods can effect on
characteristics of the mesenchymal cells from human Wharton’s Jelly. These cells
are able to expand in culture with various media and growth factors and differentiate
into neural cells.
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Abstrak Tesis Untuk Dikemukakan Kepada Senat Universiti Putra Malaysia Sebagai Memenuhi Keperluan Untuk Ijazah Doktor Falsafah
KAJIAN IN VITRO MENGENAI PERBAGAI FAKTOR DALAM PEMENCILAN, PERKEMBANGAN, DAN PEMBEZAAN SEL INDUK
MESENKIMAL DARI TALI PUSAT MANUSIA
Oleh
PARVIN SALEHINEJAD
June 2012
Pengerusi: Noorjahan Banu Binti Mohammed Alitheen, PhD Fakulti: Bioteknologi dan Sains Biomolekul Whaton Jeli (WJ) dari tali pusat adalah sumber yang kaya dengan sel induk
mesenkimal (MSCs) dan mempunyai pelbagai potensi terapeutik. Sel induk
mesenkimal dari tali pusat manusia (hUCMCs) boleh diasingkan dengan pelbagai
kaedah, dan boleh berkembang dan dibezakan menjadi neuron, astrocyte dan
oligodendrocyte secara in vitro.
Dalam kajian pertama, empat kaedah untuk mengasingkan hUCMCs telah
dibandingkan. Tiga daripada kaedah itu berdasarkan penggunaan koktel enzim yang
mengandungi kolagenase, hyaluronidase dan trypsin (CHT), kolagenase / trypsin
(CT) atau trypsin sahaja (TRP) dan selebihnya adalah berdasarkan kaedah kultur
eksplan (Exp). Dalam kajian kedua, kemampuan media DMEM / F12, α-
MEM/Glutamax (α -MEM/GL dan juga peranan faktor pertumbuhan dalam
perkembangan hUCMCs dikaji. Akhir sekali, pemeriksaan dijalankan ke atas sel
hUCMCs untuk mengenal pasti kadar perkembangan mereka menjadi sel keturunan
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neural menggunakan kombinasi enam koktail yang dibuat daripada media (DMEM /
LG), 10% FBS, Retinoic asid (RA),sulfoxide dimetil (DMSO), EGF dan FGF.
Perbandingan antara empat kaedah pengasingan sel hUCMCs menunjukkan bahawa
lebih banyak sel dapat diasingkan dalam kumpulan CT, CHT dan TRP. Sel-sel telah
berjaya diasingkan di kumpulan TRP tetapi tidak dapat berkembang secara normal.
Analisis aliran sitometri menunjukkan bahawa CD44, CD73, CD90 dan CD105 telah
diekspreskan di atas permukaan sel dalam semua kumpulan, tetapi CD34 dan CD45
tidak diekspreskan pada keturunan hematopoietik, dalam mana-mana kumpulan. Di
samping itu, ekspresi C-kit dan OCT- 4 pada kumpulan enzim (CT dan CHT) adalah
lebih baik daripada kumpulan Exp. Kadar perkembangan sel dalam kumpulan Exp
adalah jauh lebih tinggi (P <0.001) berbanding kumpulan lain. Aktiviti fosfatase alkali
yang positif dan pembezaan kepada keturunan adipogenik dan osteogenik dapat dikesan
pada sel hUCMCs dari mana-mana kumpulan.
Ekspresi stem sel mesenkimal penanda termasuklah CD44 dan CD105 adalah sama
dalam kumpulan media DMEM/F12 dan α-MEM/GL, manakala dalam kedua-dua
kumpulan ekspresi penanda CD34 pada permukaan sel-sel hematopoietic adalah negatif.
Potensi perkembangan sel kepada sel adipogenik dan osteogenik adalah sama dalam
kedua-dua kumpulan. Penilaian kadar metabolisme sel dengan WST-1 menunjukkan
bahawa kualiti sel-sel yang berkembang dalam kumpulan media α-MEM/GL adalah
lebih tinggi (P <0.001) berbanding kumpulan DMEM/F12. Populasi masa dua kali ganda
(PDT) telah dikira pada 60 jam untuk kumpulan DMEM/F12, sementara pada kumpulan
α-MEM/GL adalah 47 jam. Perbezaan ini adalah signifikan secara statistik (P <0.001).
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Separa-kuantitatif RT-PCR telah menunjukkan bahawa TERT di dalam hUCMs/EGF
adalah 0.26 ± 0.06. Analisis kitaran sel juga menunjukkan yang EGF adalah punca
kepada pergerakan 37.34± 9.8% dan 7.71± 0.53% hUCMs kepada fasa S dan G2/M.
Selepas pendedahan FGF, 29.92± 2.55% dan 9.92± 0.24% sel telah memasuki fasa S
dan G2/M.
Induksi pembezaan neuron dengan pelbagai koktel menunjukkan bahawa koktel
mengandungi kumpulan DMEM/LG, FBS, RA, FGF dan EGF (DF/R/Fg/E)
menghasilkan ekspresi dengan peratusan yang tertinggi untuk nestin, ß-tubulin III,
neurofilament, and CNPase 69.7 ± 6%, 76 ±1.7%, 89 ±9.5%, and 16.3 ±5.2%,
masing-masing. Juga di dalam kumpulan ini dan kumpulan DF/Ds/Fg/E,
perkembangan sel yang paling tinggi diperhatikan. Selain itu, kumpulan DF/Ds/Fg/E
mempunyai peratusan ekspresi GFAP yang tertinggi ((36 ±12.5%). Sementara itu,
peratusan ekspresi NF, GFAP, dan CNPase adalah yang terendah di dalam kumpulan
DF. Peratusan ekspresi terendah nestin dan ß-tubulin III diperhatikan di dalam
kumpulan DF/Ds.
MEM/GL menyokong penumbuhan hUCMCs lebih baik daripada DMEM/F12. Di
samping itu, faktor penumbuhan juga mempengaruhi ekspresi TERT dan fasa kitaran
sel. Di dalam kes ini, EGF meningkatkan ekspresi TERT dan jumlah sel yang
memasuki fasa S/G2/M. Selain itu, kejayaan hUCMCs untuk membezak kepada
pelbagai sel neuron dan jumlah pelbagai sel neuron adalah dipengaruhi oleh agen-
agen induksi. FGF dan EGF adalah ejen pendorong yang penting untuk pembezaan
hUCMCs kepada neuron, astrocyte dan oligodendrocyte. RA juga boleh mendorong
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hUCMCs untuk membezakan kepada neuron dan oligodendrocyte, DMSO
mempunyai peranan yang penting dalam pembezaan astrocyte.
Daripada kajian ini, dapat disimpulkan bahawa teknik pengasingan yang berbeza
boleh mempengaruhi ciri-ciri sel mesenchymal daripada Wharton’s Jelly manusia.
Sel-sel ini boleh berkembang di dalam kultur dengan pelbagai jenis media dan faktor
penumbuhan, dan membeza kepada sel neuron.
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ACKNOWLEDGEMENTS
I would like to express my sincere appreciation and gratitude to my supervisor Dr.
Noorjahan Banu Binti Mohammed Alitheen under whose guidance and
supervision had provided me the opportunity and conducive environment to
complete this study. Her invaluable constructive criticism and continuous support
had built in me the confidence to undertake the laboratory investigations with
patience and optimism throughout the course my studies.
Special thanks go to the members of my supervisory committee Dr. Noureddin
Nematollahi Mahani, Prof. Dr. Abdul Manaf B Ali and Prof. Dr. Abdul Rahman
Bin Omar for their suggestions and useful deliberative discussions to make this
study more comprehensive and meaningful.
Thanks are due to Dr. Noureddin Nematollahi Mahani for extending me the use of
facilities of his laboratory. I would like to thank all the staff in the Department of
Anatomy, Faculty of Kerman Medicine for their numerous help and support during
the course my work. I am equally grateful to the staff of the Flow Cytometry Unit,
Institute of Royan, especially to Mr. Ehsan Janzamin for their kind assistance in the
Flow Cytometry analaysis.
I am immensely and forever grateful to those individuals who have, in one way or
another provided me with the courage and resilience to pave the way towards the
successful completion of this study.
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Last but not least, I wish to thank my family for their continuous moral support and
encouragement. By remaining close to me through constant contact and being there
when I need them, they made me feel as if I am at home with them even though they
are far away from me.
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I certify that a Thesis Examination Committee has met on 11 June 2012 to conduct the final examination of Parvin Salehinejad on her thesis entitled “In Vitro Study of Various Factors in Isolation, Expansion and Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord“ in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee were as follows: Mohd. Puad bin Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Muhajir bin Hamid, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Cheah Yoke Kqueen, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Alp Can, PhD Professor School of Medicine Universiti Ankara Turkey (External Examiner)
----------------------------------------------- SEOW HENG FONG, PhD
Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date: 9 August 2012
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:
Noorjahan Banu Binti Mohammed Alitheen, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)
Abdul Manaf B Ali, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
Abdul Rahman B Omar, PhD Professor Institue of Biosciences Universiti Putra Malaysia (Member) Noureddin Nematollahi Mahani , PhD Professor Faculty of Medicine Universiti Kerman Iran (Member)
----------------------------------------------- BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any degree at Universiti Putra Malaysia or at any other institutions.
------------------------------------ PARVIN SALEHINEJAD
Date: 11 June 2012
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TABLE OF CONTENTS
Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xx CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 7 2.1 Stem Cells 7
2.1.1 Classifying of Stem Cells 8 2.2 Human Umbilical Cord 14
2.2.1 Development of Umbilical Cord 14 2.2.2 Umbilical Cord Structure 15
2.2.3 Wharton`s Jelly 16 2.2.4 Wharton`s Jelly Compounds 17 2.2.5 Wharton`s Jelly Cells (hUCMCs) 22 2.2.6 hUCMCs Characteristics in Vitro 29 2.2.7 hUCMCs and Tissue Engineering 31
2.2.8 hUCMCs as Compared to the Other MSC 33 2.3 Nervous System 37
2.4 Types of Neural Cells 37 2.4.1 Neurons 38 2.4.2 Neuroglial Cells 42 2.4.3 Schwann Cells 47 2.5 Neural Diseases 48 2.5.1 Current Major Treatment Approaches for Neural Diseases 48 2.5.1.1 Cell Therapy in Neural Diseases 49
3 COMPARISON OF DIFFERENT METHODS FOR ISOLATION OF
MESENCHYMAL STEM CELLS FROM HUMAN UMBILICAL WHARTON`S JELLY 51
3.1 Introduction 51 3.2 Materials and Methods 53 3.2.1 Isolation of Human Umbilical Cord Mesenchymal Cells 53 3.2.2 Detection of Cell Viability 60
3.2.3 Cell Proliferation 60 3.2.4 Detection of Cell Activity 60
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3.2.5 Detection of MSCs Phenotype 60 3.2.6 Diagnosis of Stem Cell Identity 61 3.2.7 Diagnosis of MC Identity by Differentiation into Adipocyte 62 3.2.8 Diagnosis of MC Identity by Differentiation into Osteocyte 62 3.2.9 Statistical Analysis 63
3.3 Results 63 3.4 Discussion 76 3.5 Conclusion 86
4 THE EFFECT OF ALPHA-MEM/GLUTAMEX AND DMEM/F12 MEDIA ON EXPANSION IN HUMAN UMBILICAL CORD MESENCHYMAL CELLS 87
4.1 Introduction 87 4.2 Materials and Methods 89 4.2.1 Isolation and Culture of Human Umbilical Cord Mesenchymal Cells 89 4.2.2 Cell Activity Assay 90
4.2.3 Cell Proliferation Growth Curve 90 4.2.4 Detection of Phenotype 91 4.2.5 Adipogenic and Osteogenic Differentiation 91
4.2.6 Statistical Analysis 92 4.3 Results 92 4.4 Discussion 100 4.5 Conclusion 104 5 THE EFFECT OF OF EPIDERMAL GROWTH FACTOR AND
FIBROBLAST GROWTH FACTOR ON EXPANSION IN HUMAN UMBILICAL CORD MESENCHYMAL CELLS 105
5.1 Introduction 105 5.2 Materials and Methods 108 5.2.1 Isolation of Human Umbilical Cord Mesenchymal Cells 108
5.2.2 Culture and Treatment of the Cells with Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF) 109
5.2.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 109 5.2.4 Measurement of TERT-mRNA Expression 114 5.2.5 Detection of Cell Cycle by Flow Cytometry 114 5.2.6 Statistical Analysis 115
5.3 Results 115 5.4 Discussion 123 5.5 Conclusion 130
6 DIFFERENTIATION OF HUMAN UMBILICAL CORD
MESENCHYMAL CELLS INTO NEURALCELLS IN VITRO 131 6.1 Introduction 131 6.2 Materials and Methods 133
6.2.1 Isolation and Culture of Human Umbilical Cord Mesenchymal Cells 133
6.2.2 Preparation of Mesenchymal Stem Cells 134 6.2.3 Induction of Neural Differentiation 134 6.2.4 Immunocytochemistry 135
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