Induced hepatocytes from human induced pluripotent stem cells for liver regeneration

Abstract

Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Primary human hepatocytes (PHHs) transplantation has been applied as a bridge therapy to orthologous liver transplantation. However, due to the scarcity of donor livers, development of alternative cell sources is in urgent need. For clinical therapy, surrogate hepatocytes are indispensable to recapitulate the sophisticated hepatic functions and the proliferative capacity of PHHs. More ideally, they are also autologous to avoid the problem of immune rejection. The development of induced pluripotent stem cell (iPSC) technology enables us to obtain patient-specific stem cells from somatic cell reprogramming. In addition, the progress in stem cell-based hepatic differentiation allows the induction of primary hepatocyte-like cells from iPSCs. Thus, the induced hepatocytes (iHeps) derived from iPSCs, especially patient-specific iPSCs, show great promise in autologous liver cell therapies. This study aims at developing robust and sustainable ex vivo alternative cell sources for hepatocyte transplantation in the treatment of liver disorders. First, we demonstrated the feasibility of generating human liver chimeric mouse models by transplantation of PHHs and human iPSC-derived iHeps. PHHs showed strong, long-term repopulation capacity in mouse livers that maintained for several passages. Although lower in vivo repopulation efficiency was observed in iHeps transplantation comparing to PHHs transplantation, we observed a long-term in vivo survival of iHeps without tumorigenesis, indicating the feasibility and safety of iHeps transplantation. Thus, the results confirmed the potential of humanized mice models to serve as bio-incubators for maintaining and expanding human hepatocytes, and provide near-physiological conditions for other in vivo studies such as disease modelling and drug screening. Subsequently, we determined the therapeutic effect of iHeps transplantation for the treatment of Wilson’s disease (WD), one of the liver-related metabolic disorders due to excessive intracellular accumulation of copper. By utilizing CRISPR/Cas9 gene editing technology, we corrected the disease-related genetic mutation, ATP7B R778L, in patient-derived iPSCs and transplanted the gene-corrected iPSC-derived iHeps into the livers of WD mouse models (the immunodeficient Atp7b knockout mice). The copper exportation capacity of ATP7B protein was restored in iHeps after gene correction, and WD mouse liver injuries caused by hepatic copper accumulation was alleviated after transplantation of the ATP7B-potent iHeps. The present data demonstrated the capability of iHeps to serve as a potential cell source for autologous cell therapy for WD as well as other hepatic disorders. This proof-of-principle study provided evidence on utilizing stem cell-derived ex vivo cell sources for in vivo therapeutics. Moreover, we optimized the hepatic differentiation protocol to achieve iHeps with higher maturity and differentiation efficiency. We demonstrated that through inhibition of epithelial-mesenchymal transition (EMT) process during the maturation stage, iHeps differentiation efficiency increased and in vitro maintenance prolonged. The optimized iHeps could serve as a better cell source for hepatocyte transplantation and an improved cell model for other in vitro studies. In conclusion, we established a robust model for in vivo repopulation of human hepatocytes, including PHHs and iHeps, and demonstrated the feasibility of iPSC-derived iHeps as a potential sustainable source of hepatocytes for future therapeutic usage.