The Application Research Of Human-induced Pluripotent Stem Cell-derived MSCs On Hepatic Diseases

Aim:Human-induced pluripotent stem cell-derived MSCs(hiPSC-MSCs)are a non-tissue-derived MSCs that exhibited similar characteristics to MSC derived from tissues.They are a promising alternative source of MSCs for autologous patient-specific cell therapy.However,recent study indicate that most of the biological and therapeutic effects of MSCs are attributed to paracrine mechanisms.This study aimed to evaluate the effect of exosomes derived from human-induced pluripotent stem cell-derived MSCs(hiPSC-MSCs-Exo)on hepatic ischemia-reperfusion(I/R)injury.Meanwhile,another aim of this study is to investigate a new strategy for effective induction of hepatocyte-like cells through the differentiation of hiPSC-MSCs.The purpose of this study is to introduce hiPSC-MSCs into the treatment of liver diseases by stem cells.Methods:The hiPSC-MSCs were obtained from hiPSCs by inducing them in MSC medium.Exosomes were isolated and concentrated from conditioned medium using ultracentrifugation and ultrafiltration.HiPSC-MSCs-Exo was injected systemically via the inferior vena cava in a rat model of 70%warm hepatic I/R injury and the therapeutic effect was evaluated.Serum levels of transaminases(AST and ALT)were measured using an automatic analyzer.The expression of inflammatory factors was measured using ELISAs.Histological changes were analyzed by pathological changes and inflammatory infiltration in liver tissue.Apoptosis of hepatic cells in liver tissue were measured using TUNEL staining along with apoptotic markers.The hiPSC-MSCs were cultured in a hepatic differentiation medium containing hepatocyte growth factor(HGF),oncostatin M and dexamethasone for 3 weeks.The hepatocyte-specific markers of differentiated hiPSC-MSCs were analyzed by flow cytometry,RT-qPCR,and immunofluorescent staining.Hepatocyte functions were tested including albumin production and secretion,urea production and glycogen storage.Results:The hiPSC-MSCs were positive for CD29,CD73,and CD90,and negative for CD34,CD45,and HLA-DR" HiPSC-MSCs-Exo had a diameter of 50 to 60 nm and expressed exosomal markers.Hepatocyte necrosis and sinusoidal congestion were markedly suppressed with lower SUZUKI s score after hiPSC-MSCs-Exo administration.ALT and AST,the hepatocytes injury markers,were significantly lower than control group.Inflammatory markers like TNF-a,IL-6,and HMGBI were significantly reduced with hiPSC-MSCs-Exo administration,which indicates the exosomes protective effect.Apoptotic markers like caspase-3 and bax were significantly lower and oxidative markers like GSH and SOD were significantly higher in liver tissues in experimental group,indicating exosomes anti-apoptotic effect and anti-oxidative stress response.More than 80%of hiPSC-MSCs could be induced into hepatocyte-like cells at the end of induction.The differentiated hiPSC-MSCs expressed liver specific markers such as AFP and ALB,and exhibited some hepatocyte functions,including albumin production and secretion,urea production and glycogen storage.Conclusion:Our results demonstrated that hiPSC-MSCs-Exo alleviates hepatic I/R injury,possibly via the suppression of inflammatory responses,attenuation of oxidative stress response and the inhibition of apoptosis.Meanwhile,the second part of our results demonstrated for the first time that hiPSC-MSCs are capable of differentiating into functional hepatocyte-like cells in vitro,which display characteristics more similar to adult hepatocytes than fetal ones.