Effect of ectopic OCT4 on canine adipose tissue-derived mesenchymal stem cells

Abstract

Gene modification to enhance the proliferative capacity of mesenchymal stem cells (MSCs) is critical to increase the therapeutic potentials of regenerative medicine and cell therapy for treating a wide range of diseases. To maximize the availability of MSCs for clinical application, their proliferative capacity is essential. It was hypothesized that the effect of lentivirus-mediated overexpression of canine OCT4 (cOCT4) may have an influence on the proliferation of canine adipose tissue-derived MSCs (cATMSCs). cATMSCs were successfully transduced with an cOCT4-lentiviral vector and increased expression levels of cOCT4 were confirmed by RT-PCR and immunoblotting. To identify stem cell markers for phenotypic characterization of cOCT4- and Mock-transduced cATMSCs, cell surface antigens were evaluated by flow cytometry. Both of cOCT4- and Mock-cATMSCs showed high expression of CD29, CD44, CD73, CD90 and CD105, and the absence of CD31 and CD45 surface markers. Also, it was found that expression of CD44, CD73, CD90 and CD105 was increased relatively in cOCT4-cATMSCs compared to Mock-cATMSCs. In addition, the proliferative capacity was evaluated by a WST-1 cell proliferation assay and trypan blue exclusion. cOCT4-cATMSC showed a higher proliferative ability than that of Mock-cATMSCs. The results of cell cycle analysis showed that overexpression of cOCT4 in cATMSCs caused an increase in the proportion of cells in S and G2/M phases. Increased cyclin D1 expression in cOCT4-cATMSCs was confirmed by western blot. Moreover, the expression of hepatocyte growth factor (HGF) at mRNA and protein levels was upregulated in cOCT4-cATMSCs. The results of the current study show that lentivirus-mediated overexpression of cOCT4 increased the proliferative ability of cATMSCs and expression of HGF. This improvement using OCT4 expression in ATMSCs may be useful method to expand the population without loss of stemness.