Differentiation Of Epithelial Cells And Mesenchymal Cells Derived From Human Amnion Into Hepatocytes In Rat Injured Liver

Objectives:Cell therapy-based transplantation has become a novel and effective method for treatment of serious liver disorder.Both human amniotic epithelial cells (hAECs) and human amniotic mesenchymal cells(hAMCs) could differentiate into hepatocyte-like cells in vitro.The aim of this study is to induce hAECs and hAMCs to differentiate towards hepatocytes in vivo in order to provide an experimental evidence as a new cell resource for future cell therapy-based clinical application of liver disease.Methods:The expression of surface markers and characteristics of immunocytochemistry were examined and analyzed by FCM and immunofluorescence staining.Sixty healthy and clean grade female SD rats were administered intraperitoneal injection of D-galactosamine diluted in normal saline at a dose of 400mg/kg body weight,so as to establish liver injury model,and devided into hAECs group,hAMCs group and control group stochastically,and twenty in each group.24 hours after modeling,50μl suspension(approximately 1×10~6 cells suspended in L-DMEM) of hAECs or hAMCs were injected slow into the left,middle and right lobe respectively with micro-syringe,the control group injected equivalent L-DMEM.48h,1w,2w, 3w,4w after transplantation,three or four rats of all experimental groups were executed.At 48h,1w,livers were harvested to make paraffin section and HE staining to observe pathological changes.And at 48h,1w,2w,3w,4w, distribution and the expression of human nuclear antigen,AFP,CK18,CK19 and A1b were observed by immunofluorescence staining and immunohistochemical staining on liver frozen sections.Results:①Abundant human amniotic cells could be collected by trypsin-collagenase method;Freshly isolated hAECs are negative for CD44,but hAMCs are positive (28.99±2.43%) by FCM analysis;immunofluoresce staining showed that the expression of hAECs only CK19,and vimentin expression hAMCs only.②Compared with the control group,after hAECs or hAMCs orthotopic transplantation into injured liver 48h periportal basophilic small cells are increased,liver cell necrosis less;the structure of hepatic lobules post-transplant 7d more integrated,liver cell necrosis lighter.③hAECs in situ post-transplantation of injured liver 48h are located in liver sinusoid,lw expression of AFP,2w expressed CK18,2w and 4w expression of A1b; hAMCs in situ post-transplantation of injured liver 48h are located in hepatic lobules, 1w expression of CK19,2w expression of CK18,and also human nuclear antigen and Alb can be detected at 3w post-transplantation.Conclusions:hAECs and hAMCs in damaged liver of rats can differentiate into hepatocytes,suggesting that these two human amnion cells may have important value for cell therapy-based clinical application of severe liver injury.