Title page
Contents
ABBREVIATIONS 7
Abstract 8
1. Introduction 9
2. Materials and Methods 11
2.1. Culture of hMSCs 11
2.2. Differentiation of hMSCs 11
2.3. Construction of viral vector 12
2.4. Transduction of hMSCs with the recombinant lentiviral vector 13
2.5. Reverse transcription polymerase chain reaction 13
2.6. Quantitative RT-PCR (qRT-PCR) 14
2.7. Immunocytochemistry 15
2.8. Statistical analysis 16
3. Results 17
3.1. Characteristics of hMSCs 17
3.2. In vitro expression of PDX1 from hMSCs 17
3.3. Expression of pancreatic markers in PDX1-expressing hMSCs 18
3.4. Differentiation of hMSCs into insulin-producing cells 19
4. Discussion 20
References 23
ACKNOWLEDGEMENTS 37
Table 1. Primers sequences used in polymerase chain reactions 26
Table 2. List of primary antibodies used in hMSC cell leneage 27
Figure 1. Characterization of hMSCs used in the experiment. hMSCs were tested for the ability to differentiate in vitro to multiple lineages. 29
Figure 2. Lentiviral vector construct. 30
Figure 3. Transfection of 293T cells 31
Figure 4. Induction of insulin-producing cells of PDX1-expressing hMSCs by lentiviral vectors. 32
Figure 5. RT-PCR results of transfected hMSCs after transfection at day 7. Lane1: non-transfected hMSCs, Lane2: PDX1-expressing hMSCs, Lane3: PDX1-expressing hMSCs treated basic media. 33
Figure 6. Comparision of pancreatic-specific marker expression by qRT-PCR. 34
Figure 7. Immunofluorescence reveals PDX1-hMSCs differentiated into pancreatic-like in vitro. 35
Figure 8. Ditizone staining for insulin in PDX1-expressing hMSC cutured basic media. 36
Scheme 1. Overall strategies to generate PDX1-expressing hMSCs based on lentiviral vector system 28