Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells

Abstract

Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca(2+) . We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins.

Conclusion: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches.

Figure 1

Generation of hepatoblasts from human embryonic stem cells (hESCs). (A) Protocol to differentiate hESCs into progenitors. (B) Images showing the sequential morphological changes that occur to give a polygonal shape after 10 days of culture in appropriate conditions. (C) Immunocytochemistry showing the expression of pluripotency markers (OCT4, NANOG, TRA-1-60, SSEA4) at day 0 (Panel i) followed by the expression of definitive endoderm markers (GATA4, CXCR4, HNF3β, SOX17) at day 5 (Panel ii) and the expression of hepatic endodermal cells markers (HNF3β, HNF4α, CK19, GATA4, SOX9) at day 8 (Panel iii). At day 10, cells are positive for HNF3β, AFP, HNF6, HNF4α, CK19, and GATA4 and negative for CK7 (Panel iv). (D) Flow cytometry analysis of pluripotency marker TRA-1-81 (Panel i), definitive endoderm marker CXCR4 (Panel ii) at day 5, and hepatic endoderm/progenitor marker EpCAM expression (Panel iii and iv) respectively. Scale bars = 100 μm.

Figure 2

hESC-derived hepatoblasts differentiate into cholangiocytes. (A) Diagram summarizing our cholangiocytic differentiation protocol. hESC that had been maintained in a feeder-free condition were differentiated into hepatoblasts before passaging onto collagen I-treated wells, then induced into cholangiocytic differentiation. Cells were grown 3 days in GH and EGF then IL-6. At day 17, cells reached confluency and were replated onto collagen I-treated wells. Cells were further differentiated for 3 days in IL-6, then for 2 additional days in sodium taurocholate hydrate. (B) Cholangiocytic marker gene expression level was quantified at different timepoints of the differentiation procedure by qRT-PCR. Human intrahepatic biliary epithelial cells (HIBEC) cDNA was used as a positive control. In all histograms, the value of hESC-HB was arbitrarily set to 1. *P < 0.05; **P < 0.01. (C) RT-PCR analysis of gene expression of pluripotency marker NANOG and of the biliary markers CK7, anion exchanger 2, SALL4, NOTCH2, FOXM1B, and NCAM in hESCs, hESC-derived hepatoblasts (hESC-HB), and hESC-derived cholangiocytes (hESC-Chol). Results represent the mean ± SD of three independent experiments.

Figure 3

Hepatic cells generated from hESCs display characteristics specific to cholangiocytes. (A) Immunocytochemical analysis at day 23 of differentiation shows the expression of cholangiocyte markers SOX9, OPN, CK7, CK19, CK18, HNF1β, HNF6. CK7-positive cells were negative for hepatocytic marker HNF4α. (B) Expression of cholangiocyte-specific transporters (SCTR, CFTR, ASBT, TGR5) and of VEGF receptor 2 (KDR). Scale bars = 50 μm. (C) Flow cytometry analysis illustrates that more than 90% of cells express CFTR receptor. (D) Western blot analysis confirmed the expression of cholangiocyte transporters TGR5 and ASBT in differentiated cells. (E) Immunocytochemical analysis shows the expression of acetylated α-tubulin localized on primary cilia of cholangiocytes. Cholangiocyte nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI, shown in blue). Scale bar = 20 μm.

Figure 4

Agonists induce Ca²⁺ increase in hESC-Chol. (A) RT-PCR analysis of gene expression of receptors involved in intracellular Ca²⁺ signaling: P2RY1, AChR M3, SSTR2, INSP3R type III. (B,C) Fura-2-loaded hESC-Chol were stimulated either with acetylcholine (ACh, 1 μM), somatostatin (1 nM), or ATP (30 μM) for the times indicated by the horizontal bars. Traces have been shifted arbitrarily along the y-axis for clarity. For technical convenience, traces were interrupted during the washes (each gap lasted 5 minutes). The traces shown are representative of the Ca²⁺ signal observed in the presence of these different agonists in responding cells in four independent experiments. (D) Summary of the Ca²⁺ induction data (mean ± SEM).

Figure 5

Differentiation of HepaRG-hepatoblasts into cholangiocytes. (A) Diagram of both protocols for HepaRG cell differentiation from the progenitor stage into hepatocytes or cholangiocytes. Scale bars = 50 μm. (B) QRT-PCR analysis of the cholangiocytic marker expression GGT1, JAG1, CK19, and TGR5 and of the hepatocytic marker expression ALDOB and HNF4α, compared to HIBEC (positive control), during cholangiocytic and hepatocytic differentiation, respectively. ***P < 0.001, HepaRG-Chol versus HepaRG-HB; ^##P < 0.01, HepaRG-Chol versus HepaRG-cHep; ^###P < 0.001, HepaRG-Chol versus HepaRG-cHep. (C) Immunocytochemical analysis shows the expression of OPN, CK19, SCTR, C0-029, HNF4α, and albumin in HepaRG-HB, HepaRG-cHep, and HepaRG-Chol. Scale bars = 50 μm. (D) Immunocytochemical analysis shows the expression of acetylated α-tubulin localized on primary cilia of cholangiocytes. Cholangiocyte nuclei were visualized by staining with Hoechst (shown in blue). Scale bars = 5 μm.

Figure 6

Transcriptomic profiles of cholangiocytes derived from both hESCs and HepaRG. (A) Integrative genomic analysis of hESC-Chol and HepaRG-Chol gene signature (ANOVA P < 0.001; FC >3) (dataset 1) with normal human biliary epithelial cells (dataset 2) and immortalized human intrahepatic biliary epithelial cell line H69 (dataset 3). Dataset 2 corresponds to six samples of human normal biliary epithelial cells. Dataset 3 corresponds to three samples of the H69 cell line. The dendrogram shows a significant separation of the hepatocytic lineage from hESC/hepatoblast/cholangiocyte lineages. (B) Number of genes significantly deregulated in hESC-Chol and HepaRG-Chol (P < 0.05, FC > 2). The top upstream regulators and canonical pathways were highlighted by IPA. (C) Heat map showing the expression of key genes in hESC-Chol and/or HepaRG-Chol. Up-regulation is represented by red shading; down-regulation is represented by green shading. (D) mRNA levels of common deregulated genes (middle panel), hESC-Chol specific genes (left panel), and HepaRG-Chol specific genes (right panel) were validated by qRT-PCR. A high correlation was obtained between microarray data and qRT-PCR analysis for all the specific genes validated by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Generation of cholangiocytes from hiPSCs. (A) Immunocytochemical analysis of hiPSC-derived hepatoblasts showing the expression of hepatoblast markers AFP, HNF4α, HNF6, HNF3β, and CK19. (B) qRT-PCR analysis of the cholangiocyte markers CFTR, TGR5, and AQP1 showing a significant increase in hiPSC-Chol compared to hiPSC-HB. *P < 0.05; **P < 0.01. (C) hiPSC-Chol acquire an epithelial morphology similar to that of hESC-Chol and express cholangiocytic markers SOX9, HNF6, CK19, CK7, and CFTR, whereas HNF4α expression is repressed in CK7-positive cells. Scale bars = 50 μm. (D) Immunocytochemical analysis showing the expression of acetylated α-tubulin localized in primary cilia of cholangiocytes. Cholangiocyte nuclei were visualized by staining with DAPI (shown in blue). Scale bar = 20 μm.

Figure 8

Hepatic cells generated from hESC/hiPSC/HepaRG display functional properties specific to cholangiocytes. (A) Images showing the morphology of cysts and tubules after 2 weeks in the 3D culture system. (B) Immunocytochemical analysis showing epithelial polarity by the expression of β-catenin on basolateral membrane of the cysts and of F-actin bundles on the apical side of the cells in the lumen. (C) Confocal microscopy images show transport of cholyl-lysyl-fluorescein into the central lumen of a cyst. Scale bars = 20 μm.