Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.
Bottom Line: Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures.Furthermore, we show that the size of the NKX6-1(+) population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10), and inhibitors of bone morphogenetic protein (BMP) and hedgehog signaling pathways.Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.
Affiliation: McEwen Centre for Regenerative Medicine, Toronto, ON M5G 1L7, Canada; Toronto General Research Institute, Department of Experimental Therapeutics, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: firstname.lastname@example.org.Show MeSH
Related in: MedlinePlus
Mentions: Our current differentiation protocol optimized for the generation of the pancreatic lineages (Nostro et al., 2011) supports the development of low numbers of NKX6-1+ cells along with polyhormonal insulin-expressing cells from both H1 and INSGFP/w human embryonic stem cells (hESCs). However, these cell types emerge as distinct populations in cultures (Figures S1A and S1B), consistent with the interpretation that they represent separate lineages. Based on the observations that EGF signaling is required for branching morphogenesis during endocrine lineage development, and that both EGF-family ligands and receptors are widely expressed in the mouse and human fetal pancreas (Gittes, 2009; Miettinen et al., 1995; Miettinen and Heikinheimo, 1992; Miettinen et al., 2000), we speculated that this pathway could be instrumental in the generation of hESC-derived NKX6-1-expressing pancreatic progenitors. To test this hypothesis, we generated hESC-derived pancreatic endoderm using a modified version of our previously described protocol (Nostro et al., 2011) and then cultured the cells for an additional 10 days with different combinations of EGF, NOGGIN, and nicotinamide to specify the NKX6-1 progenitor fate (Figure 1A). We then analyzed the populations for the presence of NKX6-1+ cells. NOGGIN was included because several studies have demonstrated that BMP signaling is dispensable for normal pancreatic development (Bardeesy et al., 2006; Wandzioch and Zaret, 2009), and nicotinamide was tested because it has been shown to promote endocrine differentiation from human fetal explants (Otonkoski et al., 1993). To be able to monitor NKX6-1 expression quantitatively, we used an hESC reporter line in which the GFP cDNA had been targeted to the NKX6-1 locus (A.H., A.G.E., and E.G.S., unpublished data).
Affiliation: McEwen Centre for Regenerative Medicine, Toronto, ON M5G 1L7, Canada; Toronto General Research Institute, Department of Experimental Therapeutics, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: email@example.com.