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
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Mentions: The efficient generation of functional cell types from hPSCs in vitro is dependent on precise manipulation of the appropriate signaling pathways at key developmental stages within the lineage of interest. Efforts to identify the pathways that regulate pancreatic development in vitro are complicated by the existence of two developmental programs that give rise to distinct endocrine cell types. Our findings in this study uncover important differences in the regulation of the two programs and show that NKX6-1+ progenitors are specified within 24 hr from foregut patterned endoderm with the combination of NOGGIN, RA, and FGF10. Hedgehog signaling does not appear to play a role in this specification step. The generation of NKX6-1+ progenitors from this pancreatic endoderm is dependent on EGF and nicotinamide signaling (Figure 5). In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with stage 3 factors and is dependent on inhibition of the hedgehog pathway. Expansion of the derivative lineages is not dependent on the addition of exogenous EGF and nicotinamide (Figure 5). By exploiting these differences, it is now possible to design differentiation strategies that selectively promote the generation of populations of NKX6-1+ progenitors, with minimal contamination of the polyhormonal lineage.
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.