Robust expansion and functional maturation of human hepatoblasts by chemical strategy

Abstract

Background Chemically strategies to generate hepatic cells from human pluripotent stem cells (hPSCs) for the potential clinical application have been improved. However, producing high quality and large quantities of hepatic cells remain challenging, especially in terms of step-wise efficacy and cost-effective production requires more improvements. Methods Here, we systematically evaluated chemical compounds for hepatoblast (HB) expansion and maturation to establish a robust, cost-effective, and reproducible methodology for self-renewal HBs and functional hepatocyte-like cell (HLC) production. Results The established chemical cocktail could enable HBs to proliferate nearly 3000 folds within 3 weeks with preserved bipotency. Moreover, those expanded HBs could be further efficiently differentiated into homogenous HLCs which displayed typical morphologic features and functionality as mature hepatocytes including hepatocyte identity marker expression and key functional activities such as cytochrome P450 metabolism activities and urea secretion. Importantly, the transplanted HBs in the injured liver of immune-defect mice differentiated as hepatocytes, engraft, and repopulate in the injured loci of the recipient liver. Conclusion Together, this chemical compound-based HLC generation method presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery application.

Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy

“…Our recent study has identi ed that Vc, FSK, and Dihexa could replace BMP4, EGF, and HGF, respectively, supporting HBs expansion and maintenance [13]. Also, we demonstrated that Vc could phosphorylate BMP downstream Smad1/5/8, consistent with the previous report that Vc serves a similar effect in cardiomyogenesis [40].…”

“…Hence expanding HBs, especially in small-molecule culture conditions to scalable expand HBs derived from hPSCs would be an ideal strategy for e cient, large-scale, and cost-effective generating hepatic cells for the potential clinical application [12,42]. According to this view, our previous study has established a robust and cost-effective small-molecule culture condition for the generation of self-renewing HBs and functional mature HLCs [13]. Thus, we then adopted the previously established culture condition to expand the small-molecule derived HBs.…”

“…For hepatic maturation, the proliferative HBs then cultured in previously established small-molecule culture conditions, including A8301, Dex, Dihexa, and NH4Cl (DNAD) [13]. After ve days of induction, differentiated cells displayed a homogenous and typical mature hepatocyte-like polygonal morphology (Fig.…”

“…Our previous study established a synergistic cocktail to regulate BMP, Wnt, Hedgehog, and other signaling pathways, creating a delicate balance for HBs expansion and bipotency maintenance [12]. Based on this, we further created a robust and cost-effective culture condition for the generation of self-renewing HBs and functional mature HLCs, using a small-molecule cocktail in a de ned culture system [13]. Further use of a small-molecule protocol to direct hPSCs differentiation into HBs, and establish a complete small-molecule formula to generate expanded HBs and functional mature HLCs e ciently would bene t many hepatic cell potential clinical applications, including cell transplantation, bio-arti cial liver, drug development, and disease modeling.…”

Functional human iPSC-derived alveolar-like cells cultured in a miniaturized 96‑Transwell air–liquid interface model

“…The hiPSC-derived AT2-like cell cultures were also analyzed regarding other endodermal phenotypes. No similarities were detected between AT2-like cells and hepatocytes (Alpha-1-Fetoprotein expression 51 , 52 ), thyroid-like cells (PAX8 expression in LPCs) or proximal airway markers (see Supplementary Figs. S5 f, S6 , S13 , online).…”