The Study Of Umbilical Cord Mesenchymal Stem Cells Differentiate Into Hepatocyte-like Cells

Objective: To explore an effective method of human umbilical cord mesenchymalstem cells (MSCs) induced into hepatocyte-like cells by isolating and inducing theMSC.Methods: Umbilical cord came from healthy puerperant. UCMSCs were isolatedby enzymatic digestion and cultured for3generation, and then the expression ofsurface antigen CD13, CD44, CD105, CD34, CD45, HLA-DR were detected by flowcytometry to identify whether the cells were the UCMSC. The passage3(P3) UCMSCswere induced by HGF, FGF-4, IL-6in different combinations: Group A, only the basicmedium; Group B, HGF and FGF-4put into the basic medium; Group C, HGF and IL-6put into the basic medium; Group D, HGF, FGF-4and IL-6put into the basic medium.The hepatocyte-like cell markers AFP and ALB were examed by immunocytochemistry.Data was presented as mean±standard deviation, and analyzed by ANOVA analysis inSPSS18.0statistical analysis software package.Results:1. The cell morphology of UCMSC: The UCMSC were adhered after48hours,and five days later the short spindle cells grew gradually into a long slender, then15days later the slender cell colony grow in a long strip or swirling, one month later cellscontacted with each other and covered the bottom of the entire culture dish.2. The identification of UCMSC: The third subcultivation (Passage3,P3) of theisolated cells were identified by flow cytometry to detect the expression of cell surfaceantigen. The expression rate of mesenchymal stem cell surface antigen of CD13, CD44and CD105was99.0%,98.4%and98.4%respectively, and the expression rate ofhematopoietic stem cell markers CD34, CD45and HLA-DR was4.6%,9.48%and5.04%, implying an negative result.3. The induction and differentiation of UCMSC 3.1The cell morphology of hepatoeyte-like cells: the third generation (P3) of theumbilical cord mesenchymal stem cells were induced by the combinations of cytokines.After3to4weeks the shape of cells changed from long spindle into the round orpolygonal form, and the cells presented discrete phenomenon, the cytoplasm appearsparticles, and some appeared binuclear.3.2The express of hepatocyte-like cells markers: Group A did not expresshepatocellular markers of ALB and AFP, whereas other groups with HGF, FGF-4andIL-6showed a positive expression of hepatocellular markers ALB and AFP. AFPexpression at the7th day after the induction in group B, C and D, Group D showed thehighest expression rate. At15th day, the expression rate reduced in all groups, but groupD also showed the highest rate. And after20days, almost no expression in each of thegroups. ALB expression at the7th day,15th day,20th day after the induction in groupB, C and D have strong expression. At7day, group B showed the highest expressionrate. At15th day,20th day, group B and D showed the highest expression rate.Conclusion:1. Human umbilical cord MSCs can be isolated and cultured successfully withenzymatic digestion method.2. Human umbilical cord MSCs could be steadily induced into the hepatocyte-likecells by the combination of HGF,FGF-4andIL-6in vitro in high efficiency.