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Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.

Nostro MC, Sarangi F, Yang C, Holland A, Elefanty AG, Stanley EG, Greiner DL, Keller G - Stem Cell Reports (2015)

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.

View Article: PubMed Central - PubMed

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: cnostro@uhnresearch.ca.

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Model of Pancreatic Specification from hPSCshPSCs can be efficiently differentiated into definitive endoderm, which can be patterned to a posterior-foregut-like population that has the potential to upregulate PDX1 expression upon treatment with RA and FGF10, and inhibition of SHH (using Sant-1 or cyclopamine) and BMP (using NOGGIN). PDX1-expressing cells have the potential to generate two distinct pancreatic lineages: (1) a multi-potent pancreatic progenitor expressing NKX6-1 with the potential to give rise to adult-like monohormonal cells, and (2) a polyhormonal population that lacks expression of NKX6-1 and the ability to generate functional beta cells, but generates glucagon+ cells (Basford et al., 2012). Our findings in this study uncover important differences in the regulation of the two programs and show that the monohormonal cell progenitor is specified within 24 hr by treatment with a combination of stage 3 agonists and the antagonists RA, FGF10, and NOGGIN, independently of hedgehog signaling, and that the generation of the NKX6-1+ progenitor population from this specified pancreatic endoderm is dependent on EGF and nicotinamide signaling. In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with RA, FGF10, and NOGGIN. Crucially, hedgehog inhibition and expansion of the derivative lineages are not dependent on the addition of exogenous EGF and nicotinamide.
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fig5: Model of Pancreatic Specification from hPSCshPSCs can be efficiently differentiated into definitive endoderm, which can be patterned to a posterior-foregut-like population that has the potential to upregulate PDX1 expression upon treatment with RA and FGF10, and inhibition of SHH (using Sant-1 or cyclopamine) and BMP (using NOGGIN). PDX1-expressing cells have the potential to generate two distinct pancreatic lineages: (1) a multi-potent pancreatic progenitor expressing NKX6-1 with the potential to give rise to adult-like monohormonal cells, and (2) a polyhormonal population that lacks expression of NKX6-1 and the ability to generate functional beta cells, but generates glucagon+ cells (Basford et al., 2012). Our findings in this study uncover important differences in the regulation of the two programs and show that the monohormonal cell progenitor is specified within 24 hr by treatment with a combination of stage 3 agonists and the antagonists RA, FGF10, and NOGGIN, independently of hedgehog signaling, and that the generation of the NKX6-1+ progenitor population from this specified pancreatic endoderm is dependent on EGF and nicotinamide signaling. In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with RA, FGF10, and NOGGIN. Crucially, hedgehog inhibition and expansion of the derivative lineages are not dependent on the addition of exogenous EGF and nicotinamide.

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.


Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.

Nostro MC, Sarangi F, Yang C, Holland A, Elefanty AG, Stanley EG, Greiner DL, Keller G - Stem Cell Reports (2015)

Model of Pancreatic Specification from hPSCshPSCs can be efficiently differentiated into definitive endoderm, which can be patterned to a posterior-foregut-like population that has the potential to upregulate PDX1 expression upon treatment with RA and FGF10, and inhibition of SHH (using Sant-1 or cyclopamine) and BMP (using NOGGIN). PDX1-expressing cells have the potential to generate two distinct pancreatic lineages: (1) a multi-potent pancreatic progenitor expressing NKX6-1 with the potential to give rise to adult-like monohormonal cells, and (2) a polyhormonal population that lacks expression of NKX6-1 and the ability to generate functional beta cells, but generates glucagon+ cells (Basford et al., 2012). Our findings in this study uncover important differences in the regulation of the two programs and show that the monohormonal cell progenitor is specified within 24 hr by treatment with a combination of stage 3 agonists and the antagonists RA, FGF10, and NOGGIN, independently of hedgehog signaling, and that the generation of the NKX6-1+ progenitor population from this specified pancreatic endoderm is dependent on EGF and nicotinamide signaling. In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with RA, FGF10, and NOGGIN. Crucially, hedgehog inhibition and expansion of the derivative lineages are not dependent on the addition of exogenous EGF and nicotinamide.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400642&req=5

fig5: Model of Pancreatic Specification from hPSCshPSCs can be efficiently differentiated into definitive endoderm, which can be patterned to a posterior-foregut-like population that has the potential to upregulate PDX1 expression upon treatment with RA and FGF10, and inhibition of SHH (using Sant-1 or cyclopamine) and BMP (using NOGGIN). PDX1-expressing cells have the potential to generate two distinct pancreatic lineages: (1) a multi-potent pancreatic progenitor expressing NKX6-1 with the potential to give rise to adult-like monohormonal cells, and (2) a polyhormonal population that lacks expression of NKX6-1 and the ability to generate functional beta cells, but generates glucagon+ cells (Basford et al., 2012). Our findings in this study uncover important differences in the regulation of the two programs and show that the monohormonal cell progenitor is specified within 24 hr by treatment with a combination of stage 3 agonists and the antagonists RA, FGF10, and NOGGIN, independently of hedgehog signaling, and that the generation of the NKX6-1+ progenitor population from this specified pancreatic endoderm is dependent on EGF and nicotinamide signaling. In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with RA, FGF10, and NOGGIN. Crucially, hedgehog inhibition and expansion of the derivative lineages are not dependent on the addition of exogenous EGF and nicotinamide.
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.

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.

View Article: PubMed Central - PubMed

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: cnostro@uhnresearch.ca.

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Related in: MedlinePlus