Title page
Contents
ABBREVIATIONS 12
PART I. INTRODUCTION 14
1. Human embryonic stem cells 15
2. Culture of Human ES Cells 15
3. Development of Culture methods 16
4. Cell-Surface Markers for Human ES Cells 17
5. Differentiation of human ES cells 18
6. From ES cells to osteoblast 18
7. Genetic manipulation of ES cells 20
REFERENCES 22
PART II. A NOVEL CULTURE TECHNIQUE FOR HUMAN EMBRYONIC STEM CELLS USING POROUS MEMBRANES 27
ABSTRACT 28
1. INTRODUCTION 29
2. MATERIALS AND METHODS 32
2.1. Culture of human embryonic stem cells (hESCs) on porous membrane 32
2.2. Pore size of porous membrane to prevent feeder cell migration 33
2.3. RNA extraction and Reverse transcription-PCR (RT-PCR) 33
2.4. Immunocytochemistry and Histology 34
2.5. Karyotype analysis 35
2.6. Scanning electron microscope (SEM) 35
2.7. Teratoma formation 36
2.8. Statistical analysis 36
3. RESULTS 37
3.1. Culture of human embryonic stem cells (hESCs) on porous membrane 37
3.2. Characterization of hESCs cultured by porous membrane technique (PMT) 38
3.3. Comparison of the PMT and the mechanical isolation technique (MIT) by RT-PCR and real time PCR 39
3.4. The morphologies of human ES cells cultured by PMT 40
3.5. Teratoma formation of human ES cells grown on porous membrane 40
4. DISCUSSION 42
5. REFERENCES 45
PART III. BONE FORMATION FROM HUMAN EMBRYONIC STEM CELL-DERIVED OSTEOGENIC CELLS IN POLY(D,L-LACTIC -CO-GLYCOLIC ACID)/HYDROXYAPATITE COMPOSITE SCAFFOLDS 56
ABSTRACT 57
1. INTRODUCTION 58
2. MATERIALS AND METHODS 61
2.1. Culture of human embryonic stem cells (hESCs) 61
2.2. Induction of osteogenic cells from hESCs by co-culture with primary bone-derived cells (PBDs) 61
2.3. Preparation of PLGA/HA scaffolds 62
2.4. Cell seeding and transplantation 63
2.5. Reverse Transcription-polymerase chain reaction (RT-PCR) 64
2.6. Real-time quantitative RT-PCR (Taqman method) 65
2.7. Characterization of OC-hESCs using histochemistry and immunocytochemistry 65
2.8. Flow cytometry 66
2.9. Histological analysis of in vivo bone formation 67
2.10. Soft X-ray analysis 67
2.11. Mineralization Analysis 68
2.12. Florescence in situ hybridization (FISH) 68
2.13. Statistical Analysis 69
3. RESULTS 70
3.1. In vitro osteogenic differentiation of hESCs by co-culture with PBDs 70
3.2. In vivo bone formation by implantation of scaffolds seeded with osteogenic cells derived from hESCs 70
3.3. Comparison of osteogenic factors by RT-PCR and real time PCR 71
3.4. Mineralization by osteogenic bone formation 72
3.5. Identification of OC-ESCs in generated bone tissue 73
4. DISCUSSION 74
5. REFERENCES 78
PART IV. OSTEOGENIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS USING LENTIVIRAL GENE DELIVERY SYSTEMS 89
ABSTRACT 90
1. INTRODUCTION 91
2. MATERIALS AND METHODS 93
2.1. Virus construction and production 93
2.2. Culture of mammalian embryonic stem cells 94
2.3. Transduction of mammalian embryonic stem cells 95
2.4. Reverse Transcription-polymerase chain reaction (RT-PCR) 95
2.5. FACS analysis 96
2.6. Characterization of EGFP-expressing human ES cells using Immunocytochemistry and Histology 97
2.7. Characterization of Cbfa1-expressing human ES cells using Immunocytochemistry, Histology, and western blotting 97
2.8. Karyotype analysis 98
2.9. Fluorescent in situ hybridization (FISH) 99
2.10. Teratoma formation 99
3. RESULTS 101
3.1. Promoter activities in mammalian ES cells by lentiviral gene delivery systems 101
3.2. Establishment and characterization of human ES cells (CHA3-EGFP) expressing stable transgene by lentiviral vector 102
3.3. Teratoma formation of transduced human ES cells (CHA3-EGFP) 103
3.4. In vitro osteogenic differentiation of Cbfa1-expressing human ES cells 103
4. DISCUSSION 105
5. REFERENCES 108
CONCLUSIONS 118
ACKNOWLEDGMENTS 120
Table 1. PCR primer sequence used in the present study 47
Table 2. Realtime-PCR primer and probe sequences. 81
Fig. 1-1. CHA3 and H9 human embryonic stem cells (hESCs) cultured using a porous membrane. 49
Fig. 1-2. Human embryonic stem cells (hESCs) cultured using a porous membrane. 50
Fig. 1-3. The cell number per colonies of (A) CHA3 and (B) H9 cultured for 5 days (#, p〉0.05). 51
Fig. 1-4. Characterization of human embryonic stem cells (hESCs) cultured on porous membranes with feeder STO cells attached to the opposite side. 52
Fig. 1-5. Comparison of mouse vimentin gene expression of the hESCs (CHA3 and H9) cultured by porous membrane technique (1-, 3- and 8-μm pore size) and conventional technique. 53
Fig. 1-6. Scanning electron microscope images of CHA3 hES cells that interact with feeder cells through 3-μm pores (A, B and C) and (D) 1-μm pore, respectively. 54
Fig. 1-7. In vivo differentiation in teratomas of CHA3 hESCs cultured by porous membrane technique. 55
Fig. 2-1. Characterization of osteogenic cells derived from human embryonic stem cells (OC-hESCs) by a co-culture system. 82
Fig. 2-2. Histochemical evaluation of osteogenic induction 4 and 8 weeks after in vivo implantation. 83
Fig. 2-3. Comparison of osteogenic-specific marker expression by RT-PCR analysis 4 and 8 weeks after in vivo implantation. 85
Fig. 2-4. Quantification of osteogenic-specific marker expression by real-time PCR 4 and 8 weeks after in vivo implantation. 86
Fig. 2-5. Bone formation and mineralization 4 and 8 weeks after in vivo implantation. 87
Fig. 2-6. FISH analysis of OC-hESCs 8 weeks after in vivo implantation. 88
Fig. 3-1. Lentiviral vector constructs. 111
Fig. 3-2. Activities of the CMV and EF-1α promoters in mammalian ES cells. 112
Fig. 3-3. Comparison of the efficiencies of CMV and EF-1α promoters in mouse and human ES cells by FACS analysis. 113
Fig. 3-4. Stable transgene expression following transduction by lentiviral vector. 114
Fig. 3-5. Teratoma formation in genetically modified human ES cells. 115
Fig. 3-6. SIN-EF-1α-Cbfa1-IRES-Venus. 116
Fig. 3-7. Characterization of osteogenic cells derived from Cbfa1-expressing human ES cells. 117
Scheme. 1. Overall strategies to culture human embryonic stem cells using porous membrane techniques. 48