Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails

Abstract

Background: The advent of human-induced pluripotent stem cells holds great promise for producing ample individualized hepatocytes. Although previous efforts have succeeded in generating hepatocytes from human pluripotent stem cells in vitro by viral-based expression of transcription factors and/or addition of growth factors during the differentiation process, the safety issue of viral transduction and high cost of cytokines would hinder the downstream applications. Recently, the use of small molecules has emerged as a powerful tool to induce cell fate transition for their superior stability, safety, cell permeability, and cost-effectiveness.

Methods: In the present study, we established a novel efficient hepatocyte differentiation strategy of human pluripotent stem cells with pure small-molecule cocktails. This method induced hepatocyte differentiation in a stepwise manner, including definitive endoderm differentiation, hepatic specification, and hepatocyte maturation within only 13 days.

Results: The differentiated hepatic-like cells were morphologically similar to hepatocytes derived from growth factor-based methods and primary hepatocytes. These cells not only expressed specific hepatic markers at the transcriptional and protein levels, but also possessed main liver functions such as albumin production, glycogen storage, cytochrome P450 activity, and indocyanine green uptake and release.

Conclusions: Highly efficient and expedited hepatic differentiation from human pluripotent stem cells could be achieved by our present novel, pure, small-molecule cocktails strategy, which provides a cost-effective platform for in vitro studies of the molecular mechanisms of human liver development and holds significant potential for future clinical applications.

Keywords: Hepatocyte differentiation; Human pluripotent stem cells; Small molecules.

Fig. 1

Optimization of concentration and duration of CHIR99021 treatment during DE induction. qRT-PCR for indicated genes using RNA lysates from human iPSCs treated with CHIR99021 at 1 μM or 3 μM for 24, 48, and 72 h during differentiation. Relevant expression of markers for pluripotency (a), DE (b), mesoderm (c) and ectoderm (d) were showed. e qRT-PCR for pluripotent markers (OCT4, NANOG), DE markers (SOX17, FOXA2), mesoderm markers (HAND1, BRA), and ectoderm markers (GAP43, ZIC1) using RNA lysates from human iPSCs exposed to CHIR99021 continuously or intermittent for 48 h. Growth factor-based method (activin A) was used for comparison

Fig. 2

Small molecules efficiently induce definitive endoderm differentiation from hPSCs. a qRT-PCR for pluripotent markers and DE markers using RNA lysates from human iPSCs treated with DMSO at day 1, CHIR99021 (3 μM) at day 2, and then basal medium without CHIR99021 at day 3. b Phase contrast photos (×200) showing morphological changes during stage I of differentiation. Scale bars = 100 μm. c Immunofluorescence of pluripotency and DE-specific markers at the end of differentiation stage I. Scale bars = 100 μm. d, e Percentage of FOXA2/SOX17- and CXCR4-positive cells at day 0 and day 3 of DE differentiation analyzed by flow cytometry. e Histogram of the FOXA2/SOX17- and CXCR4-positive cells at day 0 and day 3 of DE differentiation analyzed by flow cytometry. Undifferentiated human iPSCs were used as control. All data were presented as the mean of at least three independent experiments. The error bars represent the SD

Fig. 3

The small-molecule cocktail efficiently induces the formation of hepatic progenitors from definitive endoderm. a Phase contrast images showing morphology of cells in hepatic progenitor stage induced with small molecules (lower panel) or for 8 days with growth factors activin A, BMP-4 and FGF-4 (upper panel). b q-PCR analysis of hepatic markers (AFP, ALB, and HNF4α) and biliary markers (CK18 and CK19) RNA lysates from small-molecule cocktails or growth factors induced hepatic progenitors. Undifferentiated human iPSCs and HepG2 were used as controls. c AFP secretion of hepatic progenitors induced by small-molecule cocktails or growth factors. Undifferentiated human iPSCs and HepG2 were regarded as controls. d Immunofluorescence of HNF4α and AFP of differentiated cells induced by small-molecule cocktails or growth factors. Scale bars = 50 μm. e Percentage of HNF4α⁺AFP⁺ in immunofluorescence of hepatic progenitors generated using small-molecule cocktails or growth factors. (^*p value < 0.05)

Fig. 4

Characterization of small-molecule-induced hepatocyte-like cells. a qRT-PCR of hepatocyte markers at the endpoint of the small-molecule protocols with or without FH1 and FPH1. Undifferentiated human iPSCs and freshly isolated human primary hepatocytes (hPH) were used as controls. b Representative immunofluorescence photos of the expression of hepatocyte markers at the endpoint of the small-molecule-induced hepatocytes (SM-iHep) and growth factor-induced hepatocytes (GF-iHep). c Representative phase contrast photos (×200) of small-molecule-induced hepatocytes (SM-iHep) and growth factor-induced hepatocytes (GF-iHep) at day 13. Scale bars = 100 μm (d) Percentage of ALB⁺A1AT⁺ in immunofluorescence of hepatocyte-like cells generated using small-molecule cocktails or growth factors. (^*p value < 0.05, ^**p value < 0.01)

Fig. 5

Functional analysis of small-molecule-induced hepatocyte-like cells. a PAS staining showing glycogen storage in small-molecule- and growth factor-induced differentiated cells. Freshly isolated human primary hepatocytes (hPH) were used as the control. Scale bars = 100 μm. b Cytochrome P450 1A2 activity in small-molecule- (SM-iHep) and growth factor-induced hepatocytes (GF-iHep) after induction with omeprazole (1A2). c Western blotting for albumin expression of small-molecule-induced hepatocyte-like cells from hESCs-H1 and hiPSCs. GAPDH was used as the loading control. d Albumin secretion of the differentiated cells treated with small-molecule cocktails or growth factors. Undifferentiated human iPSCs and freshly isolated human primary hepatocytes (hPH) were used as controls. e Analysis of ICG uptake (left) and ICG release 6 h later (right) of small-molecule-induced hepatocyte-like cells. Scale bars = 100 μm. (^*p value < 0.05, ^**p value < 0.01)

Fig. 6

The established chemical differentiation protocol is also applicable to other human pluripotent stem cell lines. a qRT-PCR analysis of pluripotency and DE-specific markers in hESC-H1 differentiated cells at the endpoint of stage I. Undifferentiated hESC-H1 were used as control. b Immunofluorescence of FOXA2 and SOX17 in hESC-H1 differentiated cells at the endpoint of stage I. Scale bars = 50 μm. c Gene expression of hepatic progenitor cell markers in hESC-H1 differentiated cells at the endpoint of stage II. Undifferentiated hESC-H1 cells were regarded as control. d Immunofluorescence of AFP and HNF4α in hESC-H1-differentiated cells at the endpoint of stage II. Scale bars = 50 μm. e qRT-PCR analysis of hepatocyte markers in hESC-H1-differentiated cells at the endpoint of stage III. Undifferentiated hESC-H1 and freshly isolated human primary hepatocytes (hPH) were regarded as controls. f Immunofluorescence of A1AT and ALB in hESC-H1-differentiated cells at the endpoint of stage III. Scale bars = 50 μm

Fig. 7

Schematic diagram of the three-stage stepwise strategy to induce hPSC differentiation into hepatocytes. a, b The detailed hepatocyte differentiation protocol we developed using pure small molecules. c Representative images showing sequential morphologic changes during the differentiation process. Scale bars = 100 μm