Title page
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS 11
ABSTRACT 12
1. GENERAL BACKGROUND 13
1.1 Propose of the stem cell research 13
1.2 Important of the stem cell research 13
1.3 Properties of stem cells 14
1.3.1 Stem cells are unspecialized 14
1.3.2 Stem cells are capable of dividing for long periods 14
1.3.3 Stem cells can give rise to specialized cells 14
1.4. Growing of ESC 15
1.5. Characterization of ESC 16
1.6. Differentiation of ESC 17
1.7. Neural differentiation of ESC 17
1.7.1 EB-based protocols 18
1.7.2 Stromal feeder-mediated neural induction 19
1.7.3 Neural differentiation by default 20
2. INTRODUCTION 23
3. MATERIALS AND METHODS 24
3.1 hESC culture 24
3.2 Mitotic inactivation of STO cells 24
3.3 Differentiation of hESC into neuron cells 24
3.4 Differentiation of NPC into glial cells 25
3.5 RT-PCR analysis 25
3.6 Immunocytochemistry 26
3.7 Immunohitochemistry 28
3.8 5-Bromo-2-deoxyuridine incorporation assay 28
4. RESULTS 32
4.1 Characterization of hESC 32
4.2 Directed differentiation of hESC to neural progenitor cells : On the feeder-free condition 32
4.4 Differentiation of hESC into neuron cells 36
4.5 Differentiation of NPC into glial cells 39
5. DISCUSSION 42
REFERENCES 47
SUMMARY IN KOREAN 55
Table. 1 List of three germ layer primer sequences used in the RT-PCR 30
Table. 2 List of neural lineage primer sequences used in the RT-PCR 30
Table. 3 List of primary antibodies used in neural lineage 31
Fig. 1. Basic techniques for inducing neural differentiation in ESC in vitro. 22
Fig. 2. Characterization of hESC line. 33
Fig. 3. Experimental schemes. 34
Fig. 4. Feeder-free condition; stage I. 34
Fig. 5. Derivation of NPC; stage II. 35
Fig. 6. Derivation of NPC; stage II. 35
Fig. 7. Expansion of NPC; stage III. 37
Fig. 8. Characterization of hESC-derived NPC; stage III. 37
Fig. 9. Differentiation of NPC into neuron cells; stage IV. 38
Fig. 10. Br-dUTP assay; stage IV. 38
Fig. 11. Gene expression of differentiated cells at each stage by RT-PCR. 40
Fig. 12. Gene expression of differentiated cells at each stage by RT-PCR. 40
Fig. 13. Differentiation of NPC into glial cells; stage IV. 41