Hepatogenic Differentiation Of Human Minor Salivary Gland Epithelial Stem/Progenitor Cells

Objective1.Hepatic differentiate the human minor salivary glands epithelial stem/progenitor cells(hMSG-EpiPCs)in vitro and assess the hepatic characteristics and hepatocytes functions of the induced cells.2.Transplant these cells into acute liver damaged mice models and investigate survival,differentiation and liver repairing ability of the hMSG-EpiPCs,Methods1.hMSG-EpiPCs were isolated and cultured from human minor salivary glands tissue explants.The stemness marker expression of the cells were tested by immunofluorescence(IF)and flow cytometer(FCM).A defining culture medium was used to differentiate the cells into hepatocytes in vitrousing thick Matrigel culture condition and the liver-associated markers and hepatic functions of the induced cells were detected by IF,Real-time PCR,ICG uptake,PAS staining and CYP450 activity analysis.2.CM-Dil dye was used to label hMSG-EpiPCs.Acute liver injury mouse models were established by intraperitoneal injecting 20%CC14 and randomly divided into two groups:hMSG-EpiPCs group(n=12)and PBS group(n=12).Three normal mice were used as control group.Tissue specimens were collected separately 1 day,3 days,1 week and 2 weeks after surgery.The liver function of the mice were assessed.Human serum albumin(HSA)in the mice serum was detected.Histological analysis was performed on liver samples slides to determine the inflammatory reaction and IF identification was executed to show the survival and differentiation of the transplanted hMSG-EpiPCs.Results1.These hMSG-EpiPCs showed high regeneration ability and expressed epithelial progenitor/stem cell and other tissue stem cell markers such as CD29,CD49f,cytokeratins,ABCG2,PLET-1,salivary epithelial cell marker CD44,CD166,hTERT,thymic epithelial progenitor cell markers K5 and K8,endothelial cell marker CD31basal lamina protein laminin,the Wnt target genes LGR5 and LGR6,and embryonic stem cell(ESC)marker SOX2.The tissue immunofluorescent stains showed expression of CK19,K5,K8,and LGR5 both in the excretory duct and acinus;CK15+ and/or LGR6+ cells were located only in the excretory duct.The cells could be induced into functional hepatocytes in vitroand expressed liver-associated markers ALB,CYP3A4,AATand CK18,while not expressed AFP.ICG uptake,PAS staining and CYP450 activity analysiswas all positive.2.The hMSG-EpiPCs labeled by CM-Dil grew normally.The body weight of hMSG-EpiPCs group mice recovered faster than the PBS group,among with a lower ratio of liver weight to body weight of hMSG-EpiPCs group.Human serum albumin(HSA)could be detected in the serum of hMSG-EpiPCs group mice 2 weeks after injection of the cells.ALT,AST and TBIL of hMSG-EpiPCs group decreased significantly faster to baseline compared to PBS group.Histological analysis revealed that less diseased hepatocytes left inhMSG-EpiPCs group.IF identification of liver samples slides showed survival of hMSG-EpiPCs two weeks after treated and the transplanted cells expressed liver lineage markers AFP,ALB and CK18.Conclusion1.hMSG-EpiPCs could be induced into hepatic cells in vitro.The induced cells expressed liver-associated markers and showed hepatocytes function.2.Transplanted hMSG-EpiPCshave therapy potentialin liver regeneration of acute liver damaged mice models,and the transplanted cells participated in the repair and reconstruction of the damaged liver tissue.