Title Page
ACKNOWLEDGMENT
CONTENTS
ABBREVIATIONS 10
1. INTRODUCTION 11
2. MATERIALS AND METHODS 13
2.1. Culture of hESCs 13
2.2. Culture of PBDs and cell lines 13
2.3. Differentiation of human ESCs into osteoblastic cells 14
2.4. RNA extraction and Reverse Transcription-PCR (RT-PCR) 15
2.5. Histology and Immunocytochemistry 15
2.6. Fluorescence activated cell sorter (FACS) analysis 16
2.7. Enzyme-linked immunoassay (ELISA) 17
2.8. Statistical analysis 17
3. RESULTS 19
3.1. Characterization of hESCs 19
3.2. Induction of osteogenic differentiation by PBDs without exogenous factors 19
3.3. Expression of osteoblast-specific markers in hEBs cocultured by PBDs 25
3.4. PBDs express BMP 2 and 4, assoluble factors for osteogenic differentiation 29
4. DISCUSSION 32
REFERENCE 36
SUMMARY IN KOREAN 41
Table 1. Sequences of the PCR primers and expected size of the amplified product 18
Figure 1. Characterization of human embryonic stem cells 20
Figure 2. Osteogenic effectin coculture system according to different feeder cell lines 22
Figure 3. Osteogenic differentiation of hEBs cocultured with PBDs 23
Figure 4. Histological staining of hEBs in coculture for the presence of phosphate and calcium deposits by the Von Kossa and Alizarin Red S staining 24
Figure 5. Expression of osteogenic specific markers by hEBs cocultured with PBDs 26
Figure 6. Immunocytochemical staining of differenentiatied hEBs during coculture with PBDs 27
Figure 7. Differential expression of human Osteocalcin (OC) between hESCs and differentiatied hEBs by FACS analysis 28
Figure 8. Expressions of BMP 2 and BMP 4 in hEBs cocultured with PBDs by RT-PCR 30
Figure 9. Expressions o fBMP 2 and BMP 4 in hEBs cocultured with PBDs by quantitative immunoassay (ELISA) 31