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Merck

Generation of qualified clinical-grade functional hepatocytes from human embryonic stem cells in chemically defined conditions.

Cell death & disease (2019-10-12)
Zhongwen Li, Jun Wu, Lei Wang, Weifang Han, Juan Yu, Xin Liu, Yukai Wang, Ying Zhang, Guihai Feng, Wei Li, Glyn Nigel Stacey, Qi Gu, Baoyang Hu, Liu Wang, Qi Zhou, Jie Hao
RESUMEN

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.

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Sigma-Aldrich
Doxorrubicina hydrochloride, 98.0-102.0% (HPLC)
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Rifampicina, ≥95% (HPLC), powder or crystals
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Periodic Acid-Schiff (PAS) Staining System
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Cardiogreen, polymethine dye
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β-Naphthoflavone, ≥98%
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Anti-Alpha-1-Antitrypsin (AAT) antibody,Mouse monoclonal, clone 1C2, purified from hybridoma cell culture
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Monoclonal Anti-α-Fetoprotein (AFP) antibody produced in mouse, ascites fluid, clone C3
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Anti-TRA-1-60 Antibody, clone TRA-1-60, Alexa Fluor 488 Conjugate, clone TRA - 1-60, from mouse, ALEXA FLUOR 488