3D organoid cultivation improves the maturation and functional differentiation of cholangiocytes from human pluripotent stem cells

Abstract

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.

Keywords: cholangiocyte differentiation; cholangiopathy; epidermal growth factor receptor signaling; human pluripotent stem cells; organoids.

FIGURE 1

Characterization of human iPSCs and ESCs. (A) Morphology of human iPSCs and ESCs. Human ESC and iPSC colonies were positively stained with AP. (B) Expression fold of pluripotent genes OCT, NANOG, and SOX2 in human PSCs. Gene expression was normalized to GAPDH expression and HepG2215 cell line. (C) Immunofluorescence staining revealed positive pluripotent markers, including OCT4 and SOX2, in human iPSC and ESC. The nuclei of all cells were stained blue with DAPI. (D) Western blot analysis revealed increases in OCT4, NANOG, and SOX2 expression in human PSCs with statistically significant relative expression. (E) The bar chart shows protein quantification differences, including OCT4, NANOG, and SOX2, in human PSCs compared to HepG2215. Protein quantification was normalized to corresponding β-actin in Figure 1; Data are presented as mean ± SEM in (B,E); p values were determined using one-way ANOVA from at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; P, passage number; AP, alkaline phosphatase.

FIGURE 2

Protocol for generating human pluripotent stem cell-derived cholangiocytes from human PSCs and definitive endoderm stage characterization. (A) The protocol proceeds through the phases of definitive endoderm (DE), ventral foregut endoderm (VFE), hepatoblast (HB), cholangiocyte progenitor (CP), and mature cholangiocyte. (B) Light microscopy revealed morphological changes in each stage of differentiation with the transformation from stem cell phenotype toward an epithelial 2D of cholangiocyte progenitor cells and subsequent maturation into cholangiocytes by using 2D and 3D culture systems. 3D culture generated mature cholangiocytes with tube-like morphology. (C) Characterization of definitive endoderm (DE) stage at day 4 of differentiation. qPCR revealed higher expression of DE markers, SOX17, and GATA4 in 4-day than in 3-day cultures. (D) Immunofluorescence staining revealed that at day 4 of differentiation, cells expressed DE marker SOX17. (E) Flow cytometry revealed that 99.4% of cells were EpCAM⁺ and 95.2% of cells were CXCR4⁺ at day 4 of differentiation. Furthermore, 84.1% of cells were both EpCAM⁺ and CXCR4⁺. Data are presented as mean ± SEM in (C); p values were determined using one-way ANOVA from at least three independent experiments; *p < 0.05, **p < 0.01.

FIGURE 3

Characterization of hepatoblast (HB) stage at day 13 of differentiation. (A) Immunofluorescence staining revealed that HBs expressed HB marker AFP. (B) Flow cytometry revealed that 85.5% of cells were AFP⁺ at the end of the HB stage. (C) qPCR revealed that the expression of HB marker AFP had significantly increased, and endoderm markers expression, including SOX17, CLDN6, and GATA4, had significantly decreased at the HB stage. Data are presented as mean ± SEM in (C); p values were determined using independent t-test from at least three independent experiments; *p < 0.05.

FIGURE 4

Characterization of cholangiocyte organoids from human PSCs at day 28 of differentiation. (A) Comparison of core biliary markers (SOX9, CFTR), intrahepatic (JAG1, YAP1), and extrahepatic (AQP1) cholangiocyte markers in cholangiocyte organoids (COs) and cholangiocyte progenitors (CPs). (B) COs were stained to detect core cholangiocyte markers CK19 and CFTR. Positive results were obtained, and COs had a tube-like form. Additional image illustrations of small- and large cholangiocyte organoids were shown. (C) Immunofluorescence signal quantification of CFTR/DAPI ratio revealed that cells generated using a 3D culture system had a higher percentage of CFTR-positive cells. Data are presented as mean ± SEM in (A,C); p values were determined using an independent t-test from at least three independent experiments on (A) and two independent experiments on (C). Data are presented as percentage ±SEM of CFTR/DAPI positive cells in (C). Positive cell quantification was performed using ImageJ (Schneider et al., 2012; Shihan et al., 2021); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Comparison of mature cholangiocytes generated using 2D and 3D culture systems. (A) qPCR revealed that cholangiocytes generated using 3D culture system yielded higher expression of mature cholangiocyte markers and region-specific markers for cholangiocytes. (B) Immunofluorescence staining revealed differences between mature cholangiocytes generated using 2D versus 3D culture systems. (C) Functional analysis using rhodamine 123 revealed an accumulation of rhodamine inside the cholangiocyte cyst and an absence of accumulation in the verapamil group. The difference in fluorescence intensity was significantly higher. (E) Functional analysis using Forskolin-induced swelling revealed the percentage of the organoid area had increased after treatment with 10 µM of Forskolin. Data are presented as mean ± SEM in (A,D); p values were determined using independent t-test from at least three independent experiments; Data presented on the (E) were calculated by measuring the longest diameter of the organoids from a single experiment; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Characterization of COs under EGFR signaling conditions. (A) qPCR revealed that the EGF group had higher expression of overall core biliary markers and region-specific markers compared to the EGF-absence group. (B) Evaluation of COs using immunofluorescence staining for CFTR and CK19 revealed tube-like morphology in the EGF group. Data are presented as mean ± SEM in (A) from two independent experiments; *p < 0.05.

FIGURE 7

A comprehensive illustration is provided in the summary figure, depicting the differentiation stages of human pluripotent stem cells (PSCs) into mature cholangiocytes. Additionally, the figure highlights the advantages of both 2D and 3D organoid culture systems in facilitating the differentiation.