Cultivation And Hepatic Differentiation Of Human Mesenchymal Stem Cells Derived From Bone Marrow In Vitro

Objective:To isolate,cultivate and cryopreserve human bone marrow mesenchymal stem cells(MSCs),and to explore whether hMSCs can be induced to develop into hepatocyte-like cells under appropriate condition in vitro.Methods:Utilizing Ficoll step gradient density centrifugation combined with plastic adherence,we isolated,cultivated and expanded the hMSCs.We then detected the cell surface markers at passage 3-5 by Flow cytometric analysis.The cells were cultured in osteogenic differentiation medium,then cell morphology was observed,alkaline phosphatase(AKP) staining was used for identification of cell type.Also hMSCs were differentiated into hepatocyte-like cells induced by HGF and FGF-4.To identify the characters of differentiated cells,morphological changes of hMSCs were observed,the levels of glycogen storage were analyzed by periodic acid-Schiff(PAS) staining of induced hMSCs,expressions of albumin(ALB),alpha-fetoprotein(AFP) and andcytokeratin 18(CK18)were detected with Immunofluorescence technique and RT-PCR technique.RESULTS:hMSCs obtained after isolation and cultivation were homogeneous in cell morphology with good vitality,and were fibroblast-like.By Flow cytometric analysis, hMSCs at passage 3-5 expressed highly the antigens of CD29,CD105,but not the antigens of CD34.After osteogenic induction of 3 weeks,alkaline phosphatase(AKP) staining was positive;After induced by HGF and FGF-4,the spindle shaped MSCs become round shaped gradully and resembled hcpatocyte-like cells.During the cultivation,AFP,CK18 and Alb were detected,PAS staining was positive.Conclusion:Cultured and expanded human mesenchymal stem cells were homogeneous in cellular morphology,with Flow cytometric analysis,purity of hMSCs is high. Short-term cryopreserved hMSCs maintain high vitaliy;Our differentiation media, including HGF and FGF-4,are effective in differentiating bone marrow mesenchymal stem cells into hepatocyte-like cells,which express liver specific markers including ALB,AFP,(CK18).Hepatocytes glycogen storage was determined by PAS staining. These characteristics may serve as a source of cells for liver tissue engineering.