Isolation And Identification Of Human Adipose Tissue-derived Mesenchymal Stem Cells And Differentiation Into Hepatocyte-like Cells In Vitro

Objective To explore the method of isolation, culture and identification of humanadipose-derived stem cells(hADSCs) and induce the hADSCs into hepatocyte-like cellsin vitro.Methods Cut the adipose tissue into pieces of less than1mm3, obtained hADSCs bycollagenase1digestion centrifugation and then incubated the cells with a concentrationof1×104/cm2in the volume fraction of10%FBS low glucose DMEM medium andthen put them in an incubator for primary culture. Observed the cells morphologyunder inverted microscope; passaged the fifth-generation cells in the24-well plate, andcurved the cell growth curve based on the counted number of cells; digested thefifth-generation cells with trypsin and tested the stem cells surface antigens includingCD13,CD73,CD34,CD45and HLA-DR by flow cytometry(FCM); inoculated thefifth-generation cells in the6-well plate, and then induced the cells into adipocytes andosteocytes to identify their multiple differentiation potential. Inoculated hADSCs in the24-well plates, and then cultured the cells with the medium containing the factors ofActivin A, HGF, FGF, EGF, OSM, DMSO et al. Cells in the control group were culturedin the volume fraction of10%FBS low glucose DMEM medium. During the induction,detected their morphological changes. By the thirteenth, tested liver cell-related genesby RT-PCR; tested glycogen synthesis function of the hepatocyte-like cells by PASstaining; tested the expression of related proteins of liver cells by cellimmunohistochemistry.Results hADSCs obtained in the digestion centrifugation are of the uniform size and are fusiform fibroblast-like cells, their proliferation is good. Cells growth curve showedthat cells came into exponential growth phase on the fourth days, from the sixth day, thegrowth of cells became stably. Cells could survive and kept undifferentiated for a longtime in vitro. Flow cytometry showed that cells were highly expression of CD13, CD73,and the expression of CD34, CD45and HLA-DR are low; hADSCs could be inducedinto adipocytes and osteocytes. The above results indicated that the human primaryadipose stem cell lines were built successfully. After induced into hepatocyte-like cells,the human adipose-derived cells,cellular morphology gradually became polygonal orround, RT-PCR analysis indicated that the experimental group showed high expressionof liver cell-related gene, including AFP, ALB, CYP3A4, while the control groupshowed no expression or low expression. Immunohistochemistry confirmed that theexperimental group showed high expression of related proteins of liver cells, while thecontrol group showed negative or low expression.Conclusion hADSCs could be isolated and obtained using digestion centrifugation,the obtained hADSCs could be repeatedly passaged and be of long-term survival, andcould be induced into functional hepatocyte-like cells in vitro, which provideexperimental basis for ADSCs as seed cells of cell therapy, bioartificial liver andliver tissue engineering.