Differentiation Of Human Umbil Ical Cord Blood Mononuclear Cells Into Hepatocyte-like Cells Induced In Vitro

OBJECTIVE: To investigate whether or not human umbilical cord blood mononuclear cells (MNC) could be induced to differentiate into hepatocyte-like cells in vitro.METHODS: Human umbilical cord blood samples were obtained sterilely by punture umbilical vein , MNC were isolated by lymphocyte separation medium (density 1.077 g·mL^-1 ) . The surface antigen expressions of MNC were detected by flow cytomet ry. MNC were induced with DMEM medium , containing HGF alone , FGF alone , HGF + FGF or no growth factor , respectively. The markers of hepatic linear cells were examined before induction, and 7, 14, 21 and 28 day after induction by RT-PCR, immunocytochemistry and periodic acid-schiff ( PAS) in every group . AFP and Albumin mRNA were detected on 7th and 21th day af ter induction. CK-18 ,hepatocyte antigen , glycogen were detected on 28th day after induction . The markers of hepatocyte linear cells did not appear in no growth factor induction group. The positive percent s of every hepatocytic marker at the same timepoint had statistically differences between HGF + FGF and HGF alone or FGF alone ( P< 0.05).RESULTS: 1. The gradient centrifugation is fit for the separation of umbilical cord blood mononuclear cells . 2.In cytokines culture system RT-PCR analysis showed that the cells cultured 14-day and 21-day both expressed ALB, CK-18, GS, AFP mRNA, the mRNA quantity in cultured 21-day cells is 1.5 fold of that in cultured 14 -day cells;In no cytokines culture system the cells could not express these markers mRNA..Immunocytochemistry also indicated that the cells cultured 14,21-day with cytokines was positive expression, and cells cultured without cytokines was negtive. 3.The positive percent of every hepatocytic marker at the same time point had statistically difference among HGF, FGF, HGF+FGFgroups (P<0.05).CONCLUSIONS: 1. MNCs from cord blood can not be differentiated to hepatocyte-like cells in vitro without cytokines. There is no hepatocyte-like cell in fresh cord blood.2. MNCs from cord blood can be differentiated to hepatocyte-like cells in vitro culture supplemented with cytokines. With these cytokines, mononuclear cells (MNCs) from human umbilical cord blood can be differentiated to the cells with hepatocyte function. 3.The positive percent s of every hepatocytic marker at the same timepoint had statistically differences between HGF + FGF and HGF alone or FGF alone.