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

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.

Differentiation of Cells Isolated from Afterbirth Tissues into Hepatocyte-Like Cells and Their Potential Clinical Application in Liver Regeneration

“…This indicates, that pre-differentiation towards a specific cell line may result in more effective regeneration and improvement of liver function. A similar phenomenon has been observed in case of preliminary in vitro differentiation of mesenchymal stem cells derived from adipose tissue [150], hESC [151] and iPSC [152]. Some researchers remark the aspect of the involvement of mature target organ cells in the in vivo differentiation of native hAEC [115,153].…”

Cell therapy for advanced liver diseases: Repair or rebuild

“…These factors include Activin A and Wnt3a to induce hepatic endoderm formation, 85,86 bone morphogenetic protein and fibroblast growth factor to promote hepatocyte differentiation, and maturation factors hepatocyte growth factor and oncostatin-M. 87 Protocols have been adapted to produce clinical-grade hepatocyte-like cells from embryonic and inducedpluripotent stem cells (iPSCs) that are capable of liver engraftment and function in pre-clinical mouse models. 88,89 However, current protocols are unable to produce fully differentiated hepatocytes from pluripotent cells in vitro and established…”

Current Perspective: 3D Spheroid Models Utilizing Human-Based Cells for Investigating Metabolism-Dependent Drug-Induced Liver Injury

“…Currently, humanderived hepatocytes that are used for establishing these models are derived from multiple sources. These include primary human hepatocytes (PHHs), which are derived from liver patients that have undergone liver resection or biopsy (51,52); cancer cell lines, which have been isolated from liver tumors (53)(54)(55); immortalized cell lines, which are hepatocytes that have undergone genetic modification to prevent cell death and promote survival (56,57), and hepatocyte-like cells (HLCs), which are derived from pluripotent or adult stem cells (58)(59)(60)(61). Each cell type has its own advantages and disadvantages (discussed throughout), but when they are grown in vitro, they will often be phenotypically different from their in-vivo counterparts for which you are trying to make conclusions (62)(63)(64)(65)(66).…”