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, 46 (6), e101

Enhanced Proliferation and Differentiation of Oct4- And Sox2-overexpressing Human Adipose Tissue Mesenchymal Stem Cells

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Enhanced Proliferation and Differentiation of Oct4- And Sox2-overexpressing Human Adipose Tissue Mesenchymal Stem Cells

Sei-Myoung Han et al. Exp Mol Med.

Abstract

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.

Figures

Figure 1
Figure 1
Expression analysis of Oct4 and Sox2 in Oct4/Sox2-ATMSCs. (a) In RT–PCR analysis, the mRNA expression levels of Oct4 and Sox2 in Oct4/Sox2-ATMSCs were significantly higher than those of RFP-ATMSCs at 24 h post-transfection. Band densities in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of Oct4 and Sox2 expression normalized to GAPDH mRNA levels. (b) Western blots show high levels of Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs. Band densities in western blot analysis were evaluated semi-quantitatively by densitometry. Results are the ratio of Oct4 and Sox2 expression normalized to beta-actin protein levels. Data are representative of three independent experiments, with similar results. Data are expressed as the means+s.d. **P<0.01 compared with the control (RFP-ATMSCs) value as determined by Student's t-test.
Figure 2
Figure 2
Immunophenotyping of RFP- and Oct4/Sox2-transfected ATMSCs. Non-transfected MSCs at passage 3, RFP-transfected ATMSCs at passage 5 and Oct4/Sox2-transfected ATMSCs at passage 5 were immunophenotyped for CD29, CD31, CD34, CD44, CD45, CD73, CD90 and CD105 by flow cytometry. The expression of surface markers of ATMSCs transduced with Oct4/Sox2 or RFP were not different from those of non-transfected ATMSCs. Data are representative of three independent experiments, with similar results.
Figure 3
Figure 3
Proliferation assay using Oct4/Sox2-ATMSCs. (a) WST-1 assay showed that Oct4/Sox2-ATMSCs have higher cell metabolic activity than RFP-ATMSCs at 1, 2 and 3 days. (b) In the trypan blue exclusion assay, viable cell numbers were increased significantly in Oct4/Sox2-ATMSCs cultured for 9 days compared with RFP-ATMSCs. Data are expressed as the mean±s.d. **P<0.01 compared with the corresponding control value as determined by Student's t-test.
Figure 4
Figure 4
Cell cycle analysis by flow cytometry, and western blot analysis of cyclin D1 in Oct4/Sox2-ATMSCs. (a) Representative flow cytometry results of RFP- and Oct4/Sox2-ATMSCs (b) The percentage of cells at each cell cycle stage as determined by flow cytometry analysis. Compared with the control (RFP-ATMSCs), cell cycle analysis showed a decrease in the proportion of Oct4/Sox2-ATMSCs in the G0/G1 phase, whereas an increase was observed in the proportion of those in the S phase. (c) The expression of cyclin D1, which is a major regulator, was increased in Oct4/Sox2-ATMSCs compared with RFP-ATMSCs. Data are expressed as the mean+s.d. **P<0.01 compared with the corresponding control value as determined by Student's t-test.
Figure 5
Figure 5
Adipogenic differentiation assay using Oct4/Sox2-ATMSCs and RT–PCR analysis for adipogenic markers. (a) Oil red O staining for lipid droplets in Oct4/Sox2-transfected ATMSCs was strong at 7, 14 and 21 days during adipogenic differentiation compared with RFP-ATMSCs. (b) RT–PCR results show a significant increase in PPARγ and lipoprotein lipase mRNA expression at 7, 14 and 21 days during adipogenic differentiation. Band densities of PPARγ in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of PPARγ expression normalized to GAPDH mRNA levels. Data are representative of three independent experiments with similar results. (1) RFP-ATMSCs, (2) Oct4/Sox2-ATMSCs.
Figure 6
Figure 6
Osteogenic differentiation assay using Oct4/Sox2-ATMSCs and RT–PCR analysis for osteogenic markers. (a) Alizarin red S staining for mineralization in Oct4/Sox2-ATMSCs was strong at 7, 14 and 21 days during osteogenic differentiation compared with RFP-ATMSCs. (b) RT–PCR results show upregulation of collagen I and osteocalcin mRNA levels in Oct4/Sox2-ATMSCs at 7, 14 and 21 days during osteogenic differentiation compared with RFP-ATMSCs. Band densities of collagen I and osteocalcin in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of collagen I and osteocalcin expression normalized to GAPDH mRNA levels. Data are representative of three independent experiments with similar results. (1) RFP-ATMSCs, (2) Oct4/Sox2-ATMSCs.

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