PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.
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.
Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.Show MeSH
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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).
Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.