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.

Keywords: Chemical cocktail; Hepatic maturation; Hepatoblast expansion; Small molecules; Stemness maintenance.

Fig. 1

Establishment of a chemical culture cocktail for HB expansion. a Schematic of the strategy to identify chemical culture cocktail for HB expansion. b Immunostaining analyses of HB-specific marker AFP and HNF4α expression after different chemical cocktails treated. Scale bars 100 μm. c Efficiency of AFP and HNF4α expression after different chemical cocktails treatment, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted. d Quantitative RT-PCR analyzed hepatic genes and Ki67 expression in chemical cocktails treated HBs. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01. e Immunostaining analyses of AFP and HNF4α expression in chemical cocktails treated HBs. Scale bars 100 μm. f Efficiency of AFP and HNF4α expression after different chemical cocktails treatment, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted

Fig. 2

The ACDFSV culture condition enables long-term self-renewal and maintenance of HBs. a Phase-contrast images of expanding HBs. Scale bar 100 μm. b Growth curve of HBs cultured in ABCEHS cocktail and ACDFSV cocktail conditions. Cell growth curves were analyzed by obtaining a cell count. Data are presented as mean ± SEM, n = 3. c Immunostaining analyses of AFP and Ki67 expression on expanded HBs (passage 10). Scale bars 50 μm. d Flow cytometric analyses of AFP and Ki67 expression in expanded HBs (passage 10). e Immunostaining analyses of HB marker proteins on expanded HBs (passage 10). Scale bars 100 μm. f Expanded HBs differentiated into ALB and E-CAD co-staining hepatocytes after mature induction. Scale bars 50 μm. g Expanded HBs differentiated into CK19 and F-actin co-staining bile duct-like structures in 3D culture. Scale bar 20 μm

Fig. 3

Establishment of a chemical induction cocktail for HLC maturation. a Schematic of screening induction condition for hepatic maturation. b The morphology of expanded HB-derived HLCs. HLCs exhibited distinct round nuclei even some has two nuclei (white arrows). Scale bar 100 μm. c Quantitative RT-PCR analyzed AFP, ALB, and A1AT expression in HLCs derived from different induction cocktails. Gene expression was normalized to HBs. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01. d Immunostaining analyses of hepatocyte markers ALB, E-CAD, and A1AT on HLCs derived from different induction cocktails. Scale bars 100 μm. e Flow cytometric analyses for ALB and E-CAD positive rate on HLCs derived from different induction cocktails

Fig. 4

Generation of functional HLCs by two-step chemical strategy. a Schematic of the two-step chemical strategy to generate functional HLCs. b Quantitative RT-PCR analyzed the genes expression levels of the urea cycle and CYP enzymes. Gene expression was normalized to HBs. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01. c Immunostaining analyses of mature hepatocyte markers (ALB, CYP3A4, and CYP2C9) on HLCs derived from two methods. Scale bars 100 μm. d Efficiency of hepatocyte markers expression, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted. e CYP450 activity assay of different origins of hepatocytes. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01. f Urea secretion among different origins of hepatocytes were analyzed. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01. g PAS staining on different origins of hepatocytes. Scale bar represents 100 μm. h ICG uptake analyses in different origins of hepatocytes. Scale bar represents 100 μm

Fig. 5

Repopulation of mice injured liver by HB transplantation. a Survival curve of mice. b Hematoxylin and eosin staining in mice liver. c Engraftment of transplanted human HBs in mice liver after 1 week transplantation, as indicated by immunostaining of human ALB (green) and Di1 (red). d Repopulation of mice liver after 4 weeks of cell transplantation, as indicated by immunostaining of human ALB (green) and Di1 (red)