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- 자료유형
- 학위논문
- 저자정보
- 발행연도
- 2015
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Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, human Oct4 and Sox2 was transfected to confer higher expansion and differentiation capabilities.
This study is composed of three chapters. In the first chapter, the Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 overexpression was verified by Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. It was also confirmed maintenance of MSC surface markers without alterations in both RFP (control) and Oct4/Sox2-ATMSCs. From these results, the successful co-transfection of Oct4 and Sox2 into ATMSCs was confirmed. It was evaluated of MSC proliferation whether overexpression of Oct4 and Sox2 confer higher expansion capabilities. WST-1 assay showed that Oct4/Sox2-ATMSCs have higher cell metabolic activity than RFP-ATMSCs at 1, 2, and 3 days. In the trypan blue exclusion assay, viable cell numbers were increased significantly in Oct4/Sox2-ATMSCs cultured for 9 days compared to RFP-ATMSCs. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the 14.38 ± 0.84% fraction of cells in S, compared to RFP-ATMSCs of 7.59 ± 0.62% (t-test, p<0.01). Increased levels of cyclin D1 as 1.3 fold were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Therefore, MSCs transduced with Oct4/Sox2 have useful clinical applications because they have short culture duration, and costs and labor are reduced and could be a useful method to meet efficacy endpoints in clinical studies of MSC-based tissue engineering.
In the second chapter, it was evaluated capabilities of Oct4/Sox2-overexpressing ATMSCs differentiation at 7, 14, 21 days. The staining intensity of Oil Red O and Alizarin Red S staining for lipid droplets and mineralization in Oct4/Sox2-ATMSCs was stronger than that in RFP-ATMSCs, suggesting that Oct4/Sox2 expression promotes adipogenesis and osteogenesis of ATMSCs. The markers of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) and the markers of osteogenesis, collagen I and osteocalcin were also evaluated. The expression levels of PPARγ was increased as 5.4 fold and collagen I and osteocalcin was 1.9 and 1.7 fold, respectively, in Oct4/Sox2-ATMSCs (t-test, p<0.01). Improved adipogenic and osteogenic differentiation of Oct4/Sox2-ATMSCs suggests the use in cell-based therapies for the reconstruction of fat and bone. In addition, the high plasticity of Oct4/Sox2-ATMSCs may reflect their role as a possible benefit for regenerative cell therapy.
In the third chapter, hypothesis was proposed that Oct4 and Sox2 can increase “transdifferentiation” of ATMSCs into cells of the hepatic lineage. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers, albumin (ALB), transferrine and α-fetoprotein (AFP) were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that ALB was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of AFP was 0.8 fold compare to that of RFP-ATMSCs (t-test, p<0.01). The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was 20.1%, which is significantly higher than that of RFP-ATMSCs, 12.6% (ANOVA, p<0.01). Thus, urea production in Oct4/Sox2-ATMSCs was 2.9 mg/dl, which is significantly higher compared to that in RFP-ATMSCs was 1.6 mg/dl (ANOVA, p<0.01). This data suggest that the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were possibly functional mature hepatocytes, with enhanced capacity to store glycogen and produce urea. These results suggest that ATMSCs genetically engineered to overexpress Oct4 and Sox2 can be used to replace damaged hepatocytes in end-stage liver disease.
Taken together, Oct4 and Sox2 gene engineering could be a useful method for gaining high-quality ATMSCs with shortened culture time and the ability to meet efficacy endpoints in clinical studies of MSC-based tissue engineering. In addition, it was also demonstrated that enhanced transdifferentiation of Oct4 and Sox2 overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, it can be expected that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation. A new and useful alternative source that can enable hepatocyte regeneration or liver cell transplantation with which the limitation of liver cell donors for future use in human clinical applications as well as veterinary medicine.
This study is composed of three chapters. In the first chapter, the Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 overexpression was verified by Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. It was also confirmed maintenance of MSC surface markers without alterations in both RFP (control) and Oct4/Sox2-ATMSCs. From these results, the successful co-transfection of Oct4 and Sox2 into ATMSCs was confirmed. It was evaluated of MSC proliferation whether overexpression of Oct4 and Sox2 confer higher expansion capabilities. WST-1 assay showed that Oct4/Sox2-ATMSCs have higher cell metabolic activity than RFP-ATMSCs at 1, 2, and 3 days. In the trypan blue exclusion assay, viable cell numbers were increased significantly in Oct4/Sox2-ATMSCs cultured for 9 days compared to RFP-ATMSCs. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the 14.38 ± 0.84% fraction of cells in S, compared to RFP-ATMSCs of 7.59 ± 0.62% (t-test, p<0.01). Increased levels of cyclin D1 as 1.3 fold were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Therefore, MSCs transduced with Oct4/Sox2 have useful clinical applications because they have short culture duration, and costs and labor are reduced and could be a useful method to meet efficacy endpoints in clinical studies of MSC-based tissue engineering.
In the second chapter, it was evaluated capabilities of Oct4/Sox2-overexpressing ATMSCs differentiation at 7, 14, 21 days. The staining intensity of Oil Red O and Alizarin Red S staining for lipid droplets and mineralization in Oct4/Sox2-ATMSCs was stronger than that in RFP-ATMSCs, suggesting that Oct4/Sox2 expression promotes adipogenesis and osteogenesis of ATMSCs. The markers of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) and the markers of osteogenesis, collagen I and osteocalcin were also evaluated. The expression levels of PPARγ was increased as 5.4 fold and collagen I and osteocalcin was 1.9 and 1.7 fold, respectively, in Oct4/Sox2-ATMSCs (t-test, p<0.01). Improved adipogenic and osteogenic differentiation of Oct4/Sox2-ATMSCs suggests the use in cell-based therapies for the reconstruction of fat and bone. In addition, the high plasticity of Oct4/Sox2-ATMSCs may reflect their role as a possible benefit for regenerative cell therapy.
In the third chapter, hypothesis was proposed that Oct4 and Sox2 can increase “transdifferentiation” of ATMSCs into cells of the hepatic lineage. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers, albumin (ALB), transferrine and α-fetoprotein (AFP) were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that ALB was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of AFP was 0.8 fold compare to that of RFP-ATMSCs (t-test, p<0.01). The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was 20.1%, which is significantly higher than that of RFP-ATMSCs, 12.6% (ANOVA, p<0.01). Thus, urea production in Oct4/Sox2-ATMSCs was 2.9 mg/dl, which is significantly higher compared to that in RFP-ATMSCs was 1.6 mg/dl (ANOVA, p<0.01). This data suggest that the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were possibly functional mature hepatocytes, with enhanced capacity to store glycogen and produce urea. These results suggest that ATMSCs genetically engineered to overexpress Oct4 and Sox2 can be used to replace damaged hepatocytes in end-stage liver disease.
Taken together, Oct4 and Sox2 gene engineering could be a useful method for gaining high-quality ATMSCs with shortened culture time and the ability to meet efficacy endpoints in clinical studies of MSC-based tissue engineering. In addition, it was also demonstrated that enhanced transdifferentiation of Oct4 and Sox2 overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, it can be expected that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation. A new and useful alternative source that can enable hepatocyte regeneration or liver cell transplantation with which the limitation of liver cell donors for future use in human clinical applications as well as veterinary medicine.
목차
ABSTRACT...................................................................iCONTENTS.................................................................viLIST OF FIGURES.........................................................xLIST OF TABLES.........................................................xiiABBREVIATIONS.........................................................xiiiLITERATURE REVIEW 11. Cell therapy using adipose tissue-derived mesenchymal stem cells.............................................12. Gene engineering of mesenchymal stem cells........33. Transcriptional regulation of pluripotent-specific genes by Pou5f1 - POU domain, class 5, transcription factor 1 (Oct4) and Sry-box containing gene 2 (Sox2)......54. End- stage liver disease in the veterinary medicine .........................................................................65. Stem cell therapy for liver disease.........................8CHATER I. Enhanced proliferation of Oct4 and Sox2overexpressing human adipose tissue mesenchymal stem cells.................................................................141. Introduction........................................................142. Materials and Methods........................................16Ethics statement.........................................................16Cell cultures...............................................................16DNA plasmid preparation..............................................16Generation of Oct4/Sox2-overexpressing human ATMSCs .........................................................................17Flow cytometric analysis of cell surface marker expression in ATMSCs.................................................................17Reverse transcription-polymerase chain reaction (RT-PCR) analysis.....................................................................18Western blot analysis....................................................19Cell proliferation analysis..............................................20Cell cycle analysis......................................................21Statistical analysis.......................................................213. Results...............................................................22Cell morphology examination.......................................22Analysis of the Oct4 and Sox2 expression in ATMSCs transfected with Oct4/Sox2............................................22Immunophenotyping of RFP- and Oct4/Sox2-ATMSCs.....23Enhanced proliferative potential of Oct4/Sox2-ATMSCs.....23Acceleration of the G1 to S phase transition in Oct4/Sox2-ATMSCs....................................................................244. Discussion.........................................................25CHAPTER II. Enhanced adipogenic and osteogenic differentiation of Oct4 and Sox2 overexpressing human adipose tissue mesenchymal stem cells......................401. Introduction........................................................402. Materials and Methods........................................41Cell cultures...............................................................41Generation of Oct4/Sox2-overexpressing human ATMSCs ........................................................................41RT-PCR analysis.......................................................42Adipogenic differentiation.............................................43Osteogenic differentiation.............................................43Statistical analysis.....................................................443. Results.............................................................44Improved adipogenic and osteogenic differentiation of Oct4/Sox2-ATMSCs...................................................444. Discussion........................................................46CHAPTER III. Enhanced hepatogenic transdifferentiation of human adipose tissue mesenchymal stem cells by gene engineering with Oct4 and Sox2..................................561. Introduction......................................................562. Materials and Methods......................................58Cell cultures.............................................................58Generation of Oct4/Sox2-overexpressing human ATMSCs .......................................................................59RT-PCR analysis.......................................................59Hepatogenic differentiation............................................60Immunofluorescence...................................................61PAS staining..............................................................62Urea assay................................................................62Statistical analysis......................................................633. Results..............................................................63Hepatogenic differentiation of RFP- and Oct4/Sox2-ATMSCs ........................................................................63Functionality test of hepatocyte-like cells derived from RFP- and Oct4/Sox2-ATMSCs.............................................654. Discussion........................................................66CONCLUSIONS..........................................................80REFERENCES............................................................82ABSTRACT IN KOREAN (국문초록).............................100
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