Rapid Hepatogenic Differentiation Of Human Minor Salivary Gland Mesenchymal Stem Cells In Vitro

BackgroundAdult stem cell derived hepatocytes bring about novel strategy to the treatment of hepatopathy,in which the acquisition of seed cells and remodeling of liver function play the key role.There have been many successful reports about hepatic lineage differentiation using different kinds of adult stem cells.But low efficiency limits its clinical application.Minor salivary glands have a lot of advantages such as its abundant source,easy accessibility in clinical setting and small area damage,which makes them ideal source for adult stem cells.They have great research value and application potential.Using human minor salivary gland mesenchymal stem cells for rapid hepatic differentiation in vitro will provide theoretical basis and technical support for cell therapy of hepatopathy.Objective1.Isolate and characterize mesenchymal stem cells from human minor salivary glands,analysis their potential of stem cell proliferation and differentiation,to provide stem cell base for rapid hepatic differentiation in vitro.2.Induce hMSG-MSCs to hepatic differentiation rapidly in vitro,assess the expression of related genes and protein,and detect whether the cells induced finally have relevant functions of mature hepatocyte.Methods1.Culture human minor salivary glands tissue primarily,isolate and expande hMSG-MSCs in vitro.Cells were identified by flow cytometry,and were induced to Osteogenic fat differentiation.2.Under 2D culture environment,induce hMSG-MSCs to hepatic differentiation rapidly by using CHIR99021?HGF?FGF4.EGF through three steps in vitro.Observe the cell growth and morphological changes.Use real-time PCR and immunofluorescence to detect the expression of related phenotype and whether the cells induced finally have the relevant functions of mature hepatocyte.by PAS staining and ICG uptake.Results1.We acquired long spindled hMSG-MSCs successfuly.hMSG-MSCs showed high regeneration ability and their cell characteristics kept intact.Flow cytometry detection showed positive expressing markers CD29,CD90 and CD 166.CD117,CD45 and other hematopoietic stem cell markers were not expressed,and epithelial cell markers such as CD49f was also negative.They could be successfully differentiated into osteogeny and fat.2.Under 2D culture environment in vitro,it totally takes 6 days for hepatic differentiation.After the first day,80%to 90%cell morphology change to short spindle from long spindle,then they change into polygon after the second day,and we get hepatocyte-like cells at last.In the induction process,endoderm markers FOXA2,SOX17 and infantile hepatic cell markers AFP were decreased,mature hepatocyte markers ALB,CYP3A4 and AAT were continuously increased.Related immunofluorescence tests were positive.Both ICG uptake and PAS staining were positive.Conclusion1.Through primary culture of human small salivary gland tissue,hMSG-MSCs with strong ability of self-renewal and multi-directional differentiation can be obtained,which was ideal choice for rapid hepatic differentiation in vitro.2.hMSG-MSCs can be induced to mature and functional hepatocyte-like cells to some extent by just 6 days under 2D culture environment in vitro.