Title page
ACKNOWLEDGEMENT
Contents
Abstract 11
I. Introduction 12
II. Materials and methods 16
II. 1. Cell culture of Human ES cells 16
II. 2. Slow freezing method 16
II. 3. Vitrification method 17
II. 4. Assesment of the survivability of human ES cells and the spontaneous differentiation after cryopreservation 17
II. 5. RNA extraction and Reverse transcriptase-PCR 19
II. 6. Immunocytochemistry and histology 22
II. 7. In vitro differentiation of human ES cells 22
II. 8. Karyotype analysis 24
II. 9. Teratoma formation in NOD/SCID mice 24
II. 10. Statistical analysis 25
III. Results 26
III. 1. Vitrification using different vehicles on human ES cell 26
III. 2. Human ES cells sustained self-renewal and pluripotency in vitro after vitrification 28
III. 3. Post-thawed human ES cells is sustained during differentiation in vitro into progeny of all three germ layers 33
III. 4. Post-thawed human ES cells exhibited normal human chromosome 33
III. 5. Human ES cells differentiate three germ layers from teratoma in vivo after vitrified 33
IV. Discussion 38
V. Conclusions 41
References 42
Summary in Korean 47
List of Tables
Table 1. RT-PCR primers used in this experiment. 21
Table 2. List of primary antibodies used in human ES cell lineage. 23
Table 3. Comparison ofrecovery, survival and differentiation score after thawing between conventional and vitrification methods in human ES cells. 30
Figure 1. Differentiation of human ES cells into three germ layers. 13
Figure 2. Various vitrification vehicles used in the study. A) Open pulled straw, B) EM-grid, C) Micro-droplet 18
Figure 3.The undifferentiation criteria of human ES cells after thawing was evaluated via AP staining. Undifferentiated state (A), Partially differentiated state (B, arrow ; 20
Figure 4. Cultivation of post-thawing human ES cells. 27
Figure 5. Comparisons of efficiency for recovery and survival rate of human ES cells after thawing, via slow freezing and vitrification methods. 29
Figure 6. RT-PCR analysis of Oct-4, Nanog and GAPDH expression. M, 100bp DNA ladder marker ; lane 1, Na ve human ES cells ; lane 2, Post-thawed human ES cells ; lane 3, 31
Figure 7. Expression of stemness markers in post-thawed human ES cells using Immunofluorescence. 32
Figure 8. Differentiation of post-thawed human ES cells into progeny of all three germ layers in vitro. 34
Figure 9. Post-thawed human ES cells exhibited normal human chromosome 35
Figure 10. Teratoma formation in human ES cells in vivo after vitrification. 37