Culture And Differentiation Of Human Mesenchymal Stem Cells Into Hepatocytes In Vitro

Objective: To establish a method of isolating and culturing human bonemarrow-derived mesenchymal stem cells(MSCs) and to investigate whether MSCscan differentiate into hepatocytes by hepatocyte growth factor(HGF) and/orepidermal growth factor(EGF) in vitro. Methods: MSCs were extracted fromhuman bone marrow from the Ficoll Lymphocyte Separating Liquid (density1.077g/ml), cultured in Low-glucose Dulbecco's Modified Eagle'sMedium(L-DMEM) containing 10% fetal bovine serum(FBS). When the cellsgrowed the basic fusion, they were digested by pancreatin andethylenediaminetetraacetic acid(EDTA) and proliferated through a process ofsubculturing. The morphology of cells was observed under phase-contrastmicroscope and the growth curve was drawn by trypan blue staining method. Thecell cycle was tested by flow cytometry. In free-serum hepatocyte culture mediumswere added HGF,EGF,HGF+EGF and no growth factor respectively to inducethe differentiation progress of hMSCs at passage 3. The changes of morphologywere observed under phase-contrast microscope. Cells were collected on day 1,7,14,21,28. AFP,ALB and CK18 were tested by immunocytochemistry, glycogenstain test by Periodic acid Schiff(PAS) method. The biochemical method canmeasure the quantity of ALB and BUN in the culture mediums. Results: 1,ByFicoll density gradient centrifuge and monolayer cultured MSCs can successfullybe isolated from bone marrow blood in vitro. When inoculating, the modalities ofcells maybe round, triangle or virgulate; and began to be adhesive for 48h; andformation of colony could be formed on day 4-5; Many cells grew in clonalmanner on day 7, and the cultured cells were characterized by large spindle-shapedappearance by the time of 10 days. The basic fusion could be observed on aboutweek 2. The passaging cells were homogeneous spindle-shaped and there were nosuspending cells in the mediums on the whole. The growth curve had distinctcharacter with three periods, which were growth slowness ,logarithmicmultiplication and growth smoothness. About 86.4% of MSCs were found at G0,G1 phase by flow cytometry. 2,MSCs cultured in free-serum hepatocyte culturemediums containing HGF,EGF,HGF+EGF, some of them become round fromspindle-shaped after induced for 5 days, at the same time the cells were reducing alittle when replacing culture mediums. The cells were changed round likehepatocyte mostly on day 10. The expression of AFP increased progressively onday 7, then decreased on day 14. The expressions of ALB,CK18 and glycogenwere stronger with the cultured time longer. The positive rate of AFP,ALB,CK18and glycogen stain at the same time point had no statistically difference among thetwo groups as treated with HGF and HGF+EGF(P>0.05), but they were significantstatistically higher than another group as treated with EGF(P<0.05). And a little ofALB and BUN can be found in the culture mediums. The content of ALB andBUN at the same time point had no statistically difference among the three groupsas treated with HGF,EGF and HGF+EGF(P>0.05). The changes of inducing cellswere found in no growth factor group but expressions of hepatocytic markers werenot observed. Conclusion: MSCs can be very easy to be isolated and culturedin vitro, and have proliferative capacity. hMSCs can differentiate into the cells withhepatic phenotype and function in the presence of single HGF,single EGF andHGF+EGF, which provide us a potential seed cell source for liver tissueengineering.