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

Dianat N, Dubois-Pot-Schneider H, Steichen C, Desterke C, Leclerc P, Raveux A, Combettes L, Weber A, Corlu A, Dubart-Kupperschmitt A - Hepatology (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U972, Paul Brousse Hospital, Villejuif, France; Université Paris Sud, UMR-S 972, Villejuif, France; IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France; DHU Hepatinov, Paul Brousse Hospital, Villejuif, France.

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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.
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fig03: 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.

Mentions: Cholangiocyte differentiation was confirmed by immunostaining analysis, showing that hESC-derived cholangiocytes expressed CK7, CK19, CK18, osteopontin (OPN), a downstream target of SOX9 during normal development,21 SOX9, HNF6, and HNF1β, whereas HNF4α expression was not detected, as shown by costaining experiments with CK7/HNF4α (Fig. 3A).


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

Dianat N, Dubois-Pot-Schneider H, Steichen C, Desterke C, Leclerc P, Raveux A, Combettes L, Weber A, Corlu A, Dubart-Kupperschmitt A - Hepatology (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4315871&req=5

fig03: 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.
Mentions: Cholangiocyte differentiation was confirmed by immunostaining analysis, showing that hESC-derived cholangiocytes expressed CK7, CK19, CK18, osteopontin (OPN), a downstream target of SOX9 during normal development,21 SOX9, HNF6, and HNF1β, whereas HNF4α expression was not detected, as shown by costaining experiments with CK7/HNF4α (Fig. 3A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U972, Paul Brousse Hospital, Villejuif, France; Université Paris Sud, UMR-S 972, Villejuif, France; IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France; DHU Hepatinov, Paul Brousse Hospital, Villejuif, France.

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Related in: MedlinePlus