Functional Hepatocytes Differentiation From Human Embryonic Stem Cells Based On Gene Targeting Technology

Human hepatocytes are invaluable tools for the evaluation of drug absorption,distribution, metabolism and excretion/toxicity (ADME/Tox) ability in the liver,which has great potential for application in drug development. However, one majorchallenge for the pharmaceutical application of these cells is how to regulate andmaintain their metabolic function, which could be impaired dramatically and rapidlyby the in vitro culture process. Currently reliable tools to identify signaling pathwaysregulating the metabolic functions of human hepatocytes in vitro are still lacking. Theother obstacle is the shortage of donor human hepatocytes. Surrogates such as otherspecies hepatocytes or cell lines differentiated from human hepatocellular carcinoma,are not very representative system for recapitulating hepatocyte function and responseto xenobiotics. Embryonic stem cells (ESCs) essentially proliferate infinitely in vitroand maintain the ability to differentiate into a variety of tissue cells, includinghepatocytes. Thus, ES cells can serve as an inexhaustible cell source for hepatocyte.Cytochrome P4503A4(CYP3A4) is the major cytochrome P450present in adulthuman liver and is involved in the metabolism of over50%of therapeutic compoundscurrently in use. In this study, we report that Transcription Activation-Like EffectorNuclease (TALEN)-based gene targeting and directed hepatic differentiation of humanembryonic stem cells (hES cells) could be combined to identify candidates promotingexpression of the CYP enzyme CYP3A4, which is the most critical P450proteinexpressed in human hepatocytes and is responsible for the metabolism ofapproximately50%of commercial drugs. The CYP3A4gene was efficiently targetedusing TALEN, resulting in expression of green fluorescent protein (GFP) under thecontrol of the endogenous CYP3A4promoter. The CYP3A4:eGFP knock-in hES cellline was further differentiated into human hepatocytes in vitro, and the use of GFP asa reporter for CYP3A4enabled the identification of epidermal growth factor (EGF) as a signaling protein that efficiently promotes and maintains CYP3A4expression inhuman hepatocytes in vitro. The addition of EGF at the differentiated hepaticprogenitor stage increased the percentage of CYP3A4+hepatocytes up to40.2%invitro. The hepatocytes differentiated from CYP3A4:eGFP knock-in hES cell line weretransplanted into URG(Tet-uPA/Rag2-/-/Il2rg-/-) mice model, which has controllableAlb-uPA damage liver, after2weeks human serum ALBUMIN was detected in URGmice, which shows the derived hepatocytes’ function of liver reconstitution andhumanization.In conclusion, our study develop an intuitive, reliable tool to identify maturedand metabolic functional human hepatocytes system in vitro, use this system further toidentify EGF is a novel regulator that is critical for CYP3A4gene expression inhuman hepatocytes. And the combination of TALEN-based gene targeting anddirected hepatic differentiation of hES cells would potentially provide useful tools forgenerating human hepatocytes with stable metabolic functions in vitro.