Abstract
Hepatocytes have been successfully generated from human pluripotent stem cells (hPSCs). However, the cost-effective and clinical-grade generation of hepatocytes from hPSCs still need to be improved. In this study, we reported the production of functional hepatocytes from clinical-grade human embryonic stem cells (hESCs) under good manufacturing practice (GMP) requirements. We sequentially generated primitive streak (PS), definitive endoderm (DE), hepatoblasts and hepatocyte-like cells (HLCs) from hESCs in the different stages with completely defined reagents. During hepatoblast differentiation, dimethyl sulfoxide (DMSO), transferrin, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Vc-Mg), insulin, and sodium selenite were used instead of cytokines and FBS/KOSR. Then, hepatoblasts were differentiated into HLCs that had a typical hepatocyte morphology and possessed characteristics of mature hepatocytes, such as metabolic-related gene expression, albumin secretion, fat accumulation, glycogen storage, and inducible cytochrome P450 activity in vitro. HLCs integrated into the livers of Tet-uPA Rag2–/– Il2rg–/– (URG) mice, which partially recovered after transplantation. Furthermore, a series of biosafety-related experiments were performed to ensure future clinical applications. In conclusion, we developed a chemically defined system to generate qualified clinical-grade HLCs from hESCs under GMP conditions. HLCs have been proven to be safe and effective for treating liver failure. This efficient platform could facilitate the treatment of liver diseases using hESC-derived HLCs transplantation.
Highlights
As one of the most important organs in the body, the liver is mainly responsible for metabolism, detoxification, protein synthesis, and production of biochemicals necessary for digestion[1]
Other types of hepatocytes, hepatocyte-like cells (HLCs), which can be derived from human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, have been reported to improve liver function after transplantation into animal models with liver injuries[13,14,15,16,17,18,19,20]
We report the generation of qualified clinical-grade functional hepatocytes derived from a clinical-grade human embryonic stem cells (hESCs) cell line (Q-CTS-hESC-2) that has been generated for use in human therapy and biobank in good manufacturing practice (GMP) conditions[31]
Summary
As one of the most important organs in the body, the liver is mainly responsible for metabolism, detoxification, protein synthesis, and production of biochemicals necessary for digestion[1]. Li et al Cell Death and Disease (2019)10:763 diseases, benefited from the hepatocyte infusion either with or without liver transplantation. The use of primary or fetal hepatocytes has been restricted due to the lack of available healthy donors as well as limited cell proliferation, functional deficits, the risk of immune rejection, and the concern regarding ethical issues[11,12]. There are some concerns regarding the regeneration potentials of HLCs compared with primary or fetal hepatocytes. No clinical trials with HLCs have been conducted because of the concerns regarding whether HLCs have a comparable function to primary human hepatocytes and the questionable biosafety for HLCs21,22
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