Study On Differentiation Of Human Umbilical Cord-derive Mesenchymal Stem Cells Into Hepatocyte-like Cells In Vitro

Objective To explore the isolation and cultivation method of human umbilical cord derived mesenchymal stem cells and to investigate there biological characteristics.To discuss the methods of hepatocyte differentiation of umbilical cord mesenchymal stem cell.Methods The umbilical cord from full-term section patients were collected in sterile condition immediately upon delivery.Blood clot in umbilical arteries and veins were rinsed out by PBS,The umbilical cord was then cut into pieces of 1mm³.UC were incubated with collagenase typeâ…£(0.1%)for 60 min and then trypsin(0.125%) for 30min with gentle agitation at 37℃.The digested mixture was then passed through a filter to obtain cell suspension.Cells were plated in cell culture flask in DMEM(5%FBS)medium.Once 80-90%confluence had been reached,adherent cells were replated at 1:3 under the same culture conditions to amplify.The cell morphology,immunocytochemistry,flow cytometry,growth kinetics,cell cycle analysis and soft agarose cloning technique were determined.There was no cell clone observed in soft agarose.Prior to hepatic differentiation,sixth passage cells were plated at in twelve-well cell culture clusters,When the cells grew at 70%confluence, the hepatocyte differentiation group was in DMEM supplemented with 5ml/L FBS, 0.5μM/Ldexamethasone,50 g/L ITS,20μg/L HGF,20μg/L FGF4 and 0.61g/L nicotinamide for 10days,20μg/L OSM,0.5μM/L dexamethasone,50 g/L ITS later. Medium was changed every 3 days.The cells were collected at different time after induction,The cell morphology,immunocytochemistry,RT-PCR,glycogen staining were detected.Results mesenchycal stem cells were isolated from UC by collagenase and trypsin digestion sufficiently,adherent cells were observed after 24 hours and reached approximately 80-90%confluence after 10 days,The cells demonstrated a fibroblast-like phenotype.Immunocytochemical staining showed that the cells presented the positive signal of CD105,Vimentin and the negative signal of CD34, CD31,CD133.Flow cytometric analysis showed that the cells expressed high levers of CD29,CD49,CD105,but did not express hematopoietic lineage marker CD34.MTT assay showed cells doubling time was 22 hours,cell cycle analysis showed that 70-80%cells were in G₀/G₁ phase.The cell chromosome number was normal.After inducement the cells began to lose their sharp edges and progressively shrunk away on day 15.Immunocytochemistry analysis showed that the differentiated cells expresses AFP from day 7.CK18,CK19,OV6 were observed on day 14.Then AFP decresed gradually until day 28.The cells expressed ALB on day 21,and increased gradually on day 28.RT-PCR showed that the cell expressed AFP on day 7.Then AFP expression decreased gradually at later period of induction.The cells expressed ALB on day 21,and boosted gradually during the maturatin process. G.lycogen storage was seen on day 28,and positively stained for PAS.Conclusions The umbilical cord derived mesenchymal stem cells could be harvested by collagenase digesting method.The cells have strong proliferation capacity and are easily obtainable.They have the same immunophenotype with bone marrow mesenchymal stem cells.The umbilical cord derived mesenchymal stem cells may play an important role on the therapeutic and biotechnological study of stem cells. The umbilical cord derived mesenchymal stem cells are able to differentiate into hepatocyte-like cells under induction condition,and expresses hepatocyte superficial marker.The induced cells have hepatocyte specific functional features.MSCs may serve as a cell source for tissue engineering of liver.