Ye-Rin Coh

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells.

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, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. 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 fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.

Enhanced Hepatogenic Transdifferentiation of Human Adipose Tissue Mesenchymal Stem Cells by Gene Engineering with Oct4 and Sox2

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase “transdifferentiation” of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. 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 were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.