Title page
Acknowledgement
Contents
ABBREVIATIONS 8
Abstract 9
I. Introduction 10
II. Material and methods 13
1. Cell culture and differentiation 13
2. Virus constructs 13
3. Virus production 14
4. Reverse transcriptase-PCR (RT-PCR) 14
5. Real time-quantitative RT-PCR 15
6. Histology and Immunohistochemistry 16
7. Western blot analysis 17
8. Statistical analysis 17
III. Results 18
1. Generation of chondrogenic cells from hESCs by hSOX9 overexpression 18
2. Induction of different hEB morphologies derived from hSOX9-transfected hESCs 18
3. Expression of chondrogenic marker genes in hSOX9-transfected hESCs 19
4. Characterization of SOX9-transfected hESCs by cartilage-specific stainings 19
IV. Discussion 21
V. Conclusion 24
VI. References 25
Table 1. Realtime-PCR primer and probe sequences. 29
Table 2. List of primary antibodies used in human ES cell leneage. 30
Figure 1. DIC image of CHA 3 hESC and EBs 32
Figure 2. Vector constructs. Lentivirus vectors containing EF-1a promoter and Venus genes were used. SIN-EF-1 -IRES-α VENUS and SIN-EF-1α -hSox9-IRES-VENUS vectors. 33
Figure 3. RT-PCR and western blotting result of transfected hESCs A) transfected hESCs after transfection at day 0 and day 10. B) Western blotting of transfected hESCs with the anti-flag antibody. 34
Figure 4. Different morphologies of hSOX9-transfected hESCs by lentiviral Vectors. (A-C) Phase contrast microscopy (A, B) and fluorescence (C) images of a hEB, 14 days after generation from hESCs (200X, A, C: 400X, B). (D-F) Phase contrast (D, E)) fluorescence microscopy (F) images of a hEB, 14 days... 35
Figure 5. Expression analysis of chondrogenic marker genes in hSOX9-transfected hESCs. Expression of the chondrogenic marker genes is higher than undifferentiated and spontaneously differentiated hESCs. Lane 1: undifferentiated hESCs, Lane 2: spontaneously differentiated hESCs, Lane 3:... 36
Figure 6. Quantification of chondrogenic-specific marker expression by real-time (A) Collagen II expression, (B) Aggrecan expression. Lane 1: undifferentiated hESCs, Lane 2: spontaneously differentiated hESCs, Lane 3: SOX9-transfected hESCs (*; p〈0.05). 37
Figure 7. Histological analysis of chondrogenic cells derived from hSOX9-transfected hESCs. (A) Fluorescence microscope image of Venus-expressing hEB derived from hSOX9-transfected hESCs (X200), (B) Alcian blue staining of hSOX9-transfected hEB (X400). 38
Figure 8. Immunohistological analysis of hSOX9-transfected hEB using Collagen II antibody (X1000). Green-Collagen II, Blue-DAPI. 39
Scheme 1. Overall strategy to directly induce hESCs into chondrogenic cells by lentiviral vector system. 31