Research Of Hepatocyte-like Cells Formed From Human Umbilical Cord Blood Stem Cells In Vitro

OBJECTIVE :To induce the stem cell (SC) from human umbilical cord blood (UCB) in vitro cultured with cytokines and without cytokines respectively.And to investigate whether or not human umbilical blood stem cells could be induced to differentiate into hepatocyte-Like cells in vitro, providing a new source of cell types for therapies of hepatic diseases in the future.METHODS :UCB was collected under sterile condition and mononuclear cells (MNCs)were separated by 6% hydroxyethy1 starch (HES) and centrifugation through Ficoll. The isolated MNCs were cultured with (experimental group) or without (control group) 20μg/L recombinant human hepocyte growth factor (HGF), 10μg/L recombinant human fibroblast growth factor-4 (FGF-4) and 10μg/L recombinant human oncostatin M(OSM). The markers of hepatic linear cells including albumin(ALB), alpha-fetoprotein(AFP) ,cytokine 18(CK18) and hepatocyte antigen were examined before induction,and 7,14,21 and 28d after induction by immunocytochemistry, immunofluorescence, periodic acid-schiff(PAS) and flow cytometer (FCM) in every group.The AFP product in the medium at the same time point was measured by radiommunoassay (RIA) .RESULTS:1 Immunocytochemistry indicated that in the experimental group AFP was detected expression on day of 7 after induction, then it was not detected on day of 21 after induction. CK18, ALB, hepatocyte antigen were detected expression on day of 14, 21 after induction and the positive cells were gradually increased with time of induction extending. The markers of hepatocyte linear did not appear without cytokines (CKs) in the control group.2 PAS staining showed the cells cultured 21 day in the experimental group were positive expression; the cells cultured 28 day were strong positive expression. Glytogen did not appear in the control group.3 The ALB-positive cells by FCM analysis almost did not deteced on the day 7 after induction in the experimental group. With the time of induction prolonged, the ALB-positive cell radio was gradually increased. It was almost 80% on the 28th day. The ALB-positive cells did not appear in the control group.4 By immunofluorescence, ALB were detected expression on 14 day after induction in the experimental group,With the time of induction prolonged, ALB- positive cells still detected until 28 day;The ALB-positive cells did not appear in the control group. 5 By RIA, the level of AFP in culure medium was the highest on the 7th day in the experimental group. The low levels of AFP were detected at different time point in the control group.Conclusions :1 MNCs from umbilical cord blood can not be differentiated into hepatocyte-like cells in vitro without CKs.2 MNCs from umbilical cord blood can be differentiated into hepatocyte-like cells in vitro with CKs.3 Human umbilical cord blood cells may be an ideal source of stem cells for the treatment of the severe hepatic diseases.