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PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

Teo AK, Tsuneyoshi N, Hoon S, Tan EK, Stanton LW, Wright CV, Dunn NR - Stem Cell Reports (2015)

Bottom Line: ChIP-seq also revealed PDX1 occupancy at hepatic genes.In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes.These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.

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Directed Differentiation of hESCs into Early Pancreatic Progenitors(A) Schematic of 17-day pancreatic differentiation protocol. On day −2, HES3 cells are plated into fibronectin-coated transwell plates. Differentiation is initiated on day 0. Growth factors (activin A, BMP4, and FGF2) and small molecules (RA, Nic, and DAPT) were added at the indicated days (see the Experimental Procedures for additional details). The typical morphological changes that occur during differentiation are shown below the schematic. Scale bar represents 100 μm.(B) Kinetics of endodermal (GATA4), gut tube (HNF1B), and pancreatic progenitor (SOX9, PDX1, HNF6) marker gene expression by qPCR over the course of 17 days of differentiation. Error bars represent the SD of three biological replicates.(C) Immunostaining for FOXA2, HNF1B, SOX9, E-CADHERIN, or PDX1 on days 10 or 17 of differentiation as indicated. Scale bar represents 100 μm.(D) Quantitation of PDX1+ pancreatic progenitors by FACS on day 17.
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fig1: Directed Differentiation of hESCs into Early Pancreatic Progenitors(A) Schematic of 17-day pancreatic differentiation protocol. On day −2, HES3 cells are plated into fibronectin-coated transwell plates. Differentiation is initiated on day 0. Growth factors (activin A, BMP4, and FGF2) and small molecules (RA, Nic, and DAPT) were added at the indicated days (see the Experimental Procedures for additional details). The typical morphological changes that occur during differentiation are shown below the schematic. Scale bar represents 100 μm.(B) Kinetics of endodermal (GATA4), gut tube (HNF1B), and pancreatic progenitor (SOX9, PDX1, HNF6) marker gene expression by qPCR over the course of 17 days of differentiation. Error bars represent the SD of three biological replicates.(C) Immunostaining for FOXA2, HNF1B, SOX9, E-CADHERIN, or PDX1 on days 10 or 17 of differentiation as indicated. Scale bar represents 100 μm.(D) Quantitation of PDX1+ pancreatic progenitors by FACS on day 17.

Mentions: We previously reported the production of ePP cells from the differentiation of hESCs in adherent culture (Teo et al., 2012). These ePP cells were characterized by high levels of PDX1 expression on day 12, but quantitation by fluorescence-activated cell sorting (FACS) revealed that they numbered no more than 35% of the entire culture. We therefore explored other culture methodologies and platforms aimed at improving differentiation efficiency to ePP and discovered that PDX1+ cell numbers were increased substantially by initially plating hESC on fibronectin-coated transwell dishes and by extending retinoic acid (RA) treatment by 2 days and supplementing with FGF2, nicotinamide, and DAPT (FND) (see Figure 1A). On day 14, FND was replenished, and cultures were typically harvested on day 17 (Figure 1A). In this revised protocol, hESCs expectedly form a cobblestone-like lawn of DE cells by day 5 (Figure 1A). By day 10, distinct cell clusters emerge and shortly thereafter appear to undergo microlumen formation and fusion reminiscent of the tubulogenesis that occurs in vivo in the developing mouse pancreas (Figure 1A) (Kesavan et al., 2009; Villasenor et al., 2010). With continued differentiation, thickened ridges extend and intersect across the transwell in a honeycomb-like meshwork (Figure 1A).


PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

Teo AK, Tsuneyoshi N, Hoon S, Tan EK, Stanton LW, Wright CV, Dunn NR - Stem Cell Reports (2015)

Directed Differentiation of hESCs into Early Pancreatic Progenitors(A) Schematic of 17-day pancreatic differentiation protocol. On day −2, HES3 cells are plated into fibronectin-coated transwell plates. Differentiation is initiated on day 0. Growth factors (activin A, BMP4, and FGF2) and small molecules (RA, Nic, and DAPT) were added at the indicated days (see the Experimental Procedures for additional details). The typical morphological changes that occur during differentiation are shown below the schematic. Scale bar represents 100 μm.(B) Kinetics of endodermal (GATA4), gut tube (HNF1B), and pancreatic progenitor (SOX9, PDX1, HNF6) marker gene expression by qPCR over the course of 17 days of differentiation. Error bars represent the SD of three biological replicates.(C) Immunostaining for FOXA2, HNF1B, SOX9, E-CADHERIN, or PDX1 on days 10 or 17 of differentiation as indicated. Scale bar represents 100 μm.(D) Quantitation of PDX1+ pancreatic progenitors by FACS on day 17.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400640&req=5

fig1: Directed Differentiation of hESCs into Early Pancreatic Progenitors(A) Schematic of 17-day pancreatic differentiation protocol. On day −2, HES3 cells are plated into fibronectin-coated transwell plates. Differentiation is initiated on day 0. Growth factors (activin A, BMP4, and FGF2) and small molecules (RA, Nic, and DAPT) were added at the indicated days (see the Experimental Procedures for additional details). The typical morphological changes that occur during differentiation are shown below the schematic. Scale bar represents 100 μm.(B) Kinetics of endodermal (GATA4), gut tube (HNF1B), and pancreatic progenitor (SOX9, PDX1, HNF6) marker gene expression by qPCR over the course of 17 days of differentiation. Error bars represent the SD of three biological replicates.(C) Immunostaining for FOXA2, HNF1B, SOX9, E-CADHERIN, or PDX1 on days 10 or 17 of differentiation as indicated. Scale bar represents 100 μm.(D) Quantitation of PDX1+ pancreatic progenitors by FACS on day 17.
Mentions: We previously reported the production of ePP cells from the differentiation of hESCs in adherent culture (Teo et al., 2012). These ePP cells were characterized by high levels of PDX1 expression on day 12, but quantitation by fluorescence-activated cell sorting (FACS) revealed that they numbered no more than 35% of the entire culture. We therefore explored other culture methodologies and platforms aimed at improving differentiation efficiency to ePP and discovered that PDX1+ cell numbers were increased substantially by initially plating hESC on fibronectin-coated transwell dishes and by extending retinoic acid (RA) treatment by 2 days and supplementing with FGF2, nicotinamide, and DAPT (FND) (see Figure 1A). On day 14, FND was replenished, and cultures were typically harvested on day 17 (Figure 1A). In this revised protocol, hESCs expectedly form a cobblestone-like lawn of DE cells by day 5 (Figure 1A). By day 10, distinct cell clusters emerge and shortly thereafter appear to undergo microlumen formation and fusion reminiscent of the tubulogenesis that occurs in vivo in the developing mouse pancreas (Figure 1A) (Kesavan et al., 2009; Villasenor et al., 2010). With continued differentiation, thickened ridges extend and intersect across the transwell in a honeycomb-like meshwork (Figure 1A).

Bottom Line: ChIP-seq also revealed PDX1 occupancy at hepatic genes.In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes.These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.

Show MeSH
Related in: MedlinePlus