In vitro hepatic differentiation of oxalate-degrading human bone mesenchymal stem cells

Abstract

Objective To induce the differentiation of human bone mesenchmal stem cells with oxalate-degrading genes (hMSCs-frc-oxc) into hepatocyte-like cells in vitro.Methods The hMSCs-frc-oxc in passage 4 were cultured and induced to differentiate into hepatocyte-like cells in differentiation medium supplemented with 40 μg/L hepatocyte growth factor (HGF) and 10 μg/L fibroblast growth factors-4 (FGF-4).The morphological changes of cells were observed microscopically.The RNA and protein levels of hepatic markers [alpha fetal protein (AFP),cytokeratin-18 (CK-18) and albumin (ALB)] and oxalate-degrading genes (frc and oxc) were detected by using immunocytochemistry,Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR).L-02 cells and transfected mock-vehicle hMSCs (hMSCsvector) were used as positive and negative controls,respectively.Results The hMSCs-frc-oxc were cultured successfully in vitro.After induction,hMSCs-frc-oxc gradually transformed into polygonal cells.Real-time PCR revealed the expression of hepatic markers (AFP,ALB and CK-18) and oxalate-degrading genes (frc and oxc) in hMSCs-frc-oxc after inducting for 14 days and 28 days,whereas hMSCs cultured alone did not show liver specific gene expression.Immunocytochemistry revealed positive ALB expression in cells differentiating for 28 days.Western blotting revealed that the expression levels of AFP and CK-18 in differentiated cells were higher than in undifferentiated cells (P ＜ 0.05).The differentiated hMSCs-frc-oxc also expressed the oxalate-degrading genes products (myc-FCOAT and flag-OCOAD).Conclusion The hMSCs with oxalate-degrading genes could be induced to differentiate into hepatocyte-like cell by HGF and FGF-4 at certain concentrations and the hepatic differentiated hMSCs-frc-oxc could express the oxalate-degrading genes and their products. Key words: Bone mesenchymal stem cells ; Cell differentiation ; Hepatocyte ; Frc gene ; Oxc gene