Project description:We report on the successful generation of functional ciliated cholangoocytes from hPSCs that will provide new opportunities to study and treat diseases of the liver. Both monolayer- and organoid-derived cells are adaptable for further bioengineering approcahes to produce more complex hPSC-deived liver tissue with biliary ductal structures. Additionally, the successful engraftment of differentiated cholangiocytes in the liver of immune-imcompetent mice provides the basis for developing novel therapeutic strategies to treat cholangiopathies.
Project description:The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease. Current protocols to generate cholangiocytes from human pluripotent cells produce immature cells. Here the authors identify retinoic acid, BMP, cAMP and Rho kinase pathways as regulators of cholangiocyte maturation, and generate ciliated cholangiocytes expressing high levels of CFTR that form ductal structures in vivo.
Project description:Gene expression profile analysis allowed to identify a panel of genes and pathways characteristic of hESC-and HepaRG-derived cholangiocytes. Microarrays were conducted at day 23 and day 10 of differentiation for hESC-Chol and HepaRG-Chol respectively and compared to hESC-HB and HepaRG-HB. Five, four or three independent experiments were performed depending the experimental sample
Project description:Gene expression profile analysis allowed to identify a panel of genes and pathways characteristic of hESC-and HepaRG-derived cholangiocytes.
Project description:The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.
Project description:Transcriptional profiling of ciliating mouse tracheal epithelial cells compared to non-ciliating cells at two timepoints, ALI+4 and ALI+12, during differentiation in vitro. These cells are obtained from a transgenic mouse expressing GFP from a human FOXJ1 promoter, so that cells destined to become ciliated can be sorted by FACS based on their expression of GFP. Likewise, the control non-ciliated cells are identified by their lack of GFP expression. Ciliated cells were compared to Universal Reference RNA, and non-ciliated cells were compared to Universal Reference RNA. Two-color arrays were used to compare non-ciliated cells, or ciliated cell samples taken at 4 days or 12 days after establishment of the air-liquid interface (ALI), to a Universal Reference RNA. Ciliated cells vs. Universal Reference RNA or Non-ciliated cells vs. Universal Reference RNA. 3 biological replicates from independently grown and harvested cell cultures were performed for non-ciliated cells, 5 biological replicates were performed for a ciliated cell samples at ALI+12, and 3 biological replicates plus 2 technical replicates were performed for ciliated cell samples at ALI+4.
Project description:We established efficiently differentiation protocol of human extented pluripotent stem (EPS) cells to functional hepatocytes (EPS-Heps) by simply adding a pre-treatment step before applying the directed differentiation protocol for conventional human pluripotent stem cells. Importantly, compared to hepatocytes derived from conventional human pluripotent stem cells, EPS-Heps transcriptionally more resemble primary human hepatocytes.
Project description:Epidermis of Xenopus embryos forms a mucociliary epithelium constituted of basal, scattered, secreting and ciliated cells and is histologically similar to human airway mucociliary epithelium. We compared microRNAs signatures of epidermis of Xenopus embryos at stage 11.5 (gastrula, non ciliated epidermis) and at stage 26 (tailbud, ciliated epidermis). 2 technical replicates of a pool of 50 explants for each stage 11.5 (non ciliated) and 26 (ciliated) of Xenopus laevis development