Ex Vivo Differentiation Of Hepatocyte-like Cells Induced From Cord Blood Mononuclear Cells

[Background] With the breakthrough of the restrictive theory of germ layer the plasticity of adult stem cells has became a hot domain of studies, adult stem cells from umbilical cord blood with the characteristic of rich resource and simple collection method are potential seeding cells of multiple cells and tissues.[Objective] To investigate the feasibility of the differentiation of hepatocyte-like cells induced from cord blood mononuclear cells(MNC), cultured on Human Plasma Fibronectin with FGF4 and HGF, providing new cell source for the transplantation of hepatocyte.[Methods] Human umbilical cord blood samples were obtained sterilely by puncture umbilical vein, citrate phosphate dextrose as anticoagulant. 3%gelatine is used for the sedimentation of erythrocyte. MNC were isolated by lymphocyte separation medium(density 1.077g/ml). Mature cells were removed by the difference of average adhesion time. MNC purified were induced with DMEM medium containing HGF and FGF4 or no growth factor respectively. The expression of albumin(ALB), cytokeratinlS(CK-18) and alpha fetoprotein(AFP) were examed before induction ,and 7, 14, 21 and 28 day after induction by RT-PCR and immunocyte chemistry stain, glycogen synthesis were examed by PAS (periodic acid-schi) at different time.[Result] (1)After being induced MNC became enlarged. The enlargement became more and more obvious, with some cells aggregated with each other, which morphologically resembled hepatocytes, with big, ellipse, irregular round, polygonal and bi-/multi-nucleus and abundant cytoplasm. The morphological change was obvious in the region of high density. (2) Resembling as the group without cytokine, the expression of ALB and CK-18 was positive on the 7th,14th,21st and 28th day in the group with cytokine by immunocytochemical staining .AFP staining was positive on the 7th and 14th but negative in later differentiating period. (3) The expression of Glycogen was weakly positive on the 21st and heavy positive on the 28th day in the group with cytokine. (4) On the 7th, 14th, 21st and 28th day, AFP mRNA could be detected by RT-PCR and could not be detected in later differentiating periods. On the contrary, the mRNA of ALB and CK-18 could be detected at all time.[ConcLusion] HGF and FGF4 can induce the differentiation of human cord blood mononuclear cells with phenotypic and functional characteristics of hepatocyte and, hence human cord. blood may serve as a cell source for cell therapy against intractable liver disease.