Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC4315871&req=5

fig02: 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.

Mentions: Like NOTCH2 and SALL4, forkhead factor M1B (FOXM1B) is also reported to be critical for differentiation of precursors toward biliary epithelial cells.15 To further define the biliary commitment potential of our hepatoblasts we analyzed the expression of different biliary markers. We found that hESC-HBs expressed FOXM1B, NOTCH2, and SALL4 (Fig. 2C).


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)

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

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

fig02: 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.
Mentions: Like NOTCH2 and SALL4, forkhead factor M1B (FOXM1B) is also reported to be critical for differentiation of precursors toward biliary epithelial cells.15 To further define the biliary commitment potential of our hepatoblasts we analyzed the expression of different biliary markers. We found that hESC-HBs expressed FOXM1B, NOTCH2, and SALL4 (Fig. 2C).

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.

Show MeSH
Related in: MedlinePlus