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

Abstract

Background: Various methods have been developed to generate hepatic cells from human pluripotent stem cells (hPSCs) that rely on the combined use of multiple expensive growth factors, limiting industrial-scale production and widespread applications. Small molecules offer an attractive alternative to growth factors for producing hepatic cells since they are more economical and relatively stable.

Methods: We dissect small-molecule combinations and identify the ideal cocktails to achieve an optimally efficient and cost-effective strategy for hepatic cells differentiation, expansion, and maturation.

Results: We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling. Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification. The obtained hepatoblasts (HBs) could subsequently expand and mature into functional hepatocyte-like cells (HLCs) by the established chemical formulas. Thus, we established a stepwise strategy with complete small molecules for efficiently producing scalable HBs and functionally matured HLCs. The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo.

Conclusion: Our current small-molecule-based hepatic generation protocol presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery using.

Keywords: Hepatic differentiation; Hepatoblasts; Hepatocyte-like cells; Human pluripotent stem cells; Small molecule.

Fig. 1

CHIR and IDE1 inefficiently induced DE differentiation from hPSCs. A Schematic of the two-step chemical strategy to differentiate hPSCs into DE cells. CHIR³: 3 µM CHIR99021, CHIR¹: 1 µM CHIR99021. B Immunostaining analyzed SOX17, OCT4, and BRA expression in the differentiated cells. Scale bars 200 μm. C Overview of lineage relationships during DE differentiation from hPSCs. PS: primitive streak, PrE: primitive endoderm, MD: mesoderm, DE: definitive endoderm. D Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction. Data are presented as mean ± SEM, n = 3. E Immunostaining analyzed SOX17 and SOX7 expression in the differentiated cells. Scale bars 200 μm. F Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3

Fig. 2

Manipulating small-molecule cocktails to improve DE differentiation. A Schematic of the strategy to differentiate hPSCs into DE cells with small molecules. B Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction with different protocols. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of SOX17 and FOXA2 expression in DE cells derived from different protocols. Scale bars 200 μm. D The efficiency of SOX17 and FOXA2 expression after different cocktails treatment, determined by the FACS examination in differentiated cells

Fig. 3

Elevating endogenic TGF-β/Nodal signal to promote DE differentiation. A Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3. B Immunostaining analyzed SOX17 and BRA expression in the CILy and CIP-derived differentiated cells. Scale bars 100 μm. C Representative immunoblots of NODAL, TGF-β1, and the phosphorylation of Smad2/3 at day 2 during DE induction with different protocols. D Quantification of the protein expression levels. Data were expressed as mean ± SD, n = 3 for each group, * P < 0.05, ** P < 0.01 vs control (CI) group

Fig. 4

Small-molecule cocktail VDF directed hepatic specification. A Schematic of the strategy to identify chemical culture cocktails for HB specification. B RT-PCR analyzed AFP and HNF4α expression during hepatic specification. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of AFP and HNF4α expansion after 5 days of BF induction. Scale bars 100 μm. D RT-PCR results show hepatic genes expression in differentiated cells induced by different small molecule cocktails. Data are presented as mean ± SEM, n = 3. E Immunostaining analyses of AFP and HNF4α expression after different small molecule cocktails induced. F The 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. 5

Expansion and maintain of small-molecule-derived HBs. A A schematic description of stepwise differentiates hPSCs into HBs and HLCs. B Flow cytometric sorting of E-pCAM⁺ HBs from day 8 differentiated cells. C Phase-contrast images of expanding HBs. Scale bar 100 μm. D Flow cytometric analyses of E-pCAM and Ki67 expression in expanded HBs (passage 20). E Immunostaining analyses of HBs marker proteins on expanded HBs. Scale bars 100 μm

Fig. 6

Differentiation of proliferative HBs into functional hepatocytes. A The morphology of small-molecule protocol-derived HLCs. HLCs exhibited polygonal shapes and distinct round nuclei; even some had two nuclei. Scale bar 100 μm. B Quantitative RT-PCR analyzed the genes expression levels of ALB, the CYP and urea cycle 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, A1AT, CYP3A4, and CYP2C9) on small-molecule protocol-derived HLCs. Scale bars 100 μm. D CYP450 activity assay of HLCs. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. E Urea secretion of HLCs was analyzed. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. F PAS staining on HLCs. Scale bar represents 200 μm. G ICG uptake analyses in HLCs. Scale bar represents 200 μm. H The HLCs were stained by Alexa-Flour 488-ac-LDL. Scale bar 200 μM

Fig. 7

Repopulation of mice injured liver by HBs transplantation. A Schematic diagram depicting HBs transplantation experimental schedule in immune-deficiency mice B Survival curve of mice. C Hematoxylin and eosin staining in mice liver. Scale bar 200 μM. D, E Engraftment of transplanted human HBs in mice liver after 1 week transplantation, as indicated by immunostaining of human ALB (green) and Di1 (red). Sham mice's liver as the negative control. Scale bar 100 μM. F Detection of AST and ALT levels in mice serum at one week after HBs transplantation. Data were analyzed by 2-tailed t tests. G After 4 weeks of cell transplantation, the reproduction of mice liver is indicated by immunostaining of human ALB (green) and Di1 (red). Scale bar 100 μM. H Flow cytometric analyses human ALB-positive hepatocytes in mice liver transplanted with human HBs. I Human ALB secretion in the mice serum. Data are presented as mean ± SEM, n = 4