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 Ca2+. 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. (Hepatology 2014;60:700–714)

Take a deep breath and digest the material: organoids and biomaterials of the respiratory and digestive systems

“…Deriving biliary organoids will allow for an in vitro human model of the drug secretion system and biliary gland diseases. [102][103][104][105] For new and current drugs, it is crucial to understand the process of elimination through the biliary gland, which could help facilitate drug development and design, [106,107] and the biliary organoids could provide a system to model this process. In addition to drug secretion, biliary gland organoids derived from iPSC lines can model genetic diseases such as cystic fibrosis (CF) where the cystic fibrosis transmembrane conductance regulator (CFTR) is impaired and disrupts fluid secretion within the glands.…”

“…[86,109] These cells have been derived from hPSC and liver progenitor cell lines. [102][103][104][105]110] The liver and biliary gland are derived from the same progenitor population during development, called hepatoblasts. [86,109,111] This population has been derived from hPSCs and has the capability of becoming either hepatocytes or cholangiocytes depending on the growth factors added to the culture.…”

“…The hPSC-derived cholangiocytes can be cultured in a three-dimensional environment that consists of Matrigel and Collagen type I. [102][103][104][105] In the three-dimensional environment, the hPSC-derived cholangiocytes had proper cellular organization forming an apical-basal polarity with barrier function, similar to native cholangiocytes. [102][103][104][105]110] When hPSC-derived cholangiocytes were co-cultured with Op9 stromal cell to stimulate Notch signaling, ductal, and tubular structures formed similar to native bile ducts.…”

“…[102] Cholangiocytes derived without stromal cells did form the apical-basal polarity, but resembled a cyst-like structure rather than the native ductal structures. [103][104][105] To model the secretion system, the cell barrier with the MDR1 transporter was tested as a function of the biliary gland. [102][103][104][105]110] Thus, an intact cell barrier is crucial to the biliary gland function.…”

“…[103][104][105] To model the secretion system, the cell barrier with the MDR1 transporter was tested as a function of the biliary gland. [102][103][104][105]110] Thus, an intact cell barrier is crucial to the biliary gland function. Since the combination of Matrigel and Collagen have been used to maintain the biliary organoids, defining the matrix parameters necessary to establish the biliary organoid barrier could facilitate the development of synthetic systems that provide a consistent and reproducible screening system.…”