FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

Abstract

Growth factors are important regulators during organ development. For many vertebrates (but not humans) it is known how they contribute to the formation and expansion of PDX1-positive cells during pancreas organogenesis. Here, the effects of the fibroblast growth factors FGF2, FGF7, FGF10, and epidermal growth factor (EGF) on pancreas development in humans were assessed by using human pluripotent stem cells (hPSCs). During this, FGF2 was identified as a potent anti-pancreatic factor whereas FGF7, FGF10, and EGF increased the cell mass while retaining PDX1-positivity. FGF2 increased the expression of the anti-pancreatic factor sonic hedgehog (SHH) while suppressing PDX1 in a dose-dependent manner. Differentiating cells secreted SHH to the medium and we interrogated the cells’ secretome during differentiation to globally examine the composition of secreted signaling factors. Members of the TGF-beta-, Wnt-, and FGF-pathways were detected. FGF17 showed a suppressive anti-pancreatic effect comparable to FGF2. By inhibition of specific branches of FGF-receptor signaling, we allocated the SHH-induction by FGF2 to MEK/ERK-signaling and the anti-pancreatic effect of FGF2 to the receptor variant FGFR1c or 3c. Altogether, we report findings on the paracrine activity of differentiating hPSCs during generation of pancreatic progenitors. These observations suggest a different role for FGF2 in humans compared to animal models of pancreas organogenesis.

Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

“…This underlines the importance of this signaling pathway for differentiation protocols of hPSC into SC-derived beta cells. According to current and previous data, Wnt/beta-catenin not only prevents development of endocrine progenitors [28], but also earlier development of PDX1+ pancreatic-duodenal cells [8] and, as shown here, development into SOX9 MPCs.…”

“…FGF7 and FGF10 were less effective with regard to the absolute number of SOX9 MPCs or showed lower gene expression of typical MPC marker genes. Previously we had identified FGF2 as a repressor of early development into PDX1 pancreatic-duodenal cells [8]. In this study SOX9 MPC generation was also slightly less effective when compared to EGF.…”

“…We can show that PDX1+ pancreatic-duodenal cells described in earlier studies [7, 8], effectively differentiate into SOX9+ multipotent pancreatic progenitor cells (MPCs) with co-expression of CPA1 or NKX6.1. By use of this triple knock-in cell line we could track the conversion of SOX9 MPCs into SC-derived beta cells.…”

“…Protocol # 6, which is based on our protocol for the differentiation of hPSC into PDX1+ pancreatic-duodenal cells (Supplementary Fig. 1E) [7, 8] expanded by Stage 3 and Stage 4 media from [11] was then selected and termed 2D experimental differentiation protocol (Fig. 1B).…”

“…The SOX9 reporter cell lines showed peak SOX9/GFP2 expression after 10-13 days of differentiation using an experimental 2D differentiation protocol. This protocol is based on previous publications by our group in which we established a robust method for generating NKX6.1-/ PDX1+ pancreatic-duodenal cells [7, 8, 17] as well as adopted stage 3/stage 4 media described by Nostro and colleagues [11].…”