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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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NKX6-1 Cells Generated Using NOGGIN, EGF, and Nicotinamide Have the Potential to Generate Islet-like StructuresImmunohistochemical analysis of NKX6-1, INS, GCG, SST, PP, CK19, and TRYP expression in the graft recovered from the mammary fat pad at 6 months post-transplantation of H1 d13 differentiated cells. Scale bar, 200 μm. See also Figures S4 and S5.
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fig4: NKX6-1 Cells Generated Using NOGGIN, EGF, and Nicotinamide Have the Potential to Generate Islet-like StructuresImmunohistochemical analysis of NKX6-1, INS, GCG, SST, PP, CK19, and TRYP expression in the graft recovered from the mammary fat pad at 6 months post-transplantation of H1 d13 differentiated cells. Scale bar, 200 μm. See also Figures S4 and S5.

Mentions: To determine whether the NENA-induced population contains endocrine progenitors, we transplanted cells from d13 H1-derived cultures into the mammary fat pad of immunocompromised NSG mice as previously described (Basford et al., 2012). The grafts were harvested at 1.5, 3, 5, and 6 months post-transplantation and analyzed for the expression of NKX6-1, cytokeratin-19 (CK19), insulin (INS), C-peptide, glucagon (GCG), somatostatin (SST), and pancreatic polypeptide (PP). At the 1.5-month time point, we detected high numbers of NKX6-1+ cells and cells that expressed CK19 (Figure S4A). Low numbers of INS+, C-peptide+, and GCG+ cells were also detected at this time point. Almost all of these INS+ cells were NKX6-1+ and none co-expressed C-peptide and GCG, indicating that they represent monohormonal endocrine cells (Figure S4A). Interestingly, the INS+ cells were also CK19− but were located in close proximity to CK19+ cells (Figure S4A). Between 3 and 6 months post-transplantation, we observed islet-like structures within the grafts (Figure 4 and data not shown) that contained NKX6-1+, INS+, GCG+, SST+, and PP+ monohormonal cells. Trypsin+ and CK19+ cells were also detected at this time point, suggesting that acinar and ductal lineage cells were present in the graft. Interestingly, at this time point, fewer endocrine cells were found within the CK19+ structures.


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)

NKX6-1 Cells Generated Using NOGGIN, EGF, and Nicotinamide Have the Potential to Generate Islet-like StructuresImmunohistochemical analysis of NKX6-1, INS, GCG, SST, PP, CK19, and TRYP expression in the graft recovered from the mammary fat pad at 6 months post-transplantation of H1 d13 differentiated cells. Scale bar, 200 μm. See also Figures S4 and S5.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig4: NKX6-1 Cells Generated Using NOGGIN, EGF, and Nicotinamide Have the Potential to Generate Islet-like StructuresImmunohistochemical analysis of NKX6-1, INS, GCG, SST, PP, CK19, and TRYP expression in the graft recovered from the mammary fat pad at 6 months post-transplantation of H1 d13 differentiated cells. Scale bar, 200 μm. See also Figures S4 and S5.
Mentions: To determine whether the NENA-induced population contains endocrine progenitors, we transplanted cells from d13 H1-derived cultures into the mammary fat pad of immunocompromised NSG mice as previously described (Basford et al., 2012). The grafts were harvested at 1.5, 3, 5, and 6 months post-transplantation and analyzed for the expression of NKX6-1, cytokeratin-19 (CK19), insulin (INS), C-peptide, glucagon (GCG), somatostatin (SST), and pancreatic polypeptide (PP). At the 1.5-month time point, we detected high numbers of NKX6-1+ cells and cells that expressed CK19 (Figure S4A). Low numbers of INS+, C-peptide+, and GCG+ cells were also detected at this time point. Almost all of these INS+ cells were NKX6-1+ and none co-expressed C-peptide and GCG, indicating that they represent monohormonal endocrine cells (Figure S4A). Interestingly, the INS+ cells were also CK19− but were located in close proximity to CK19+ cells (Figure S4A). Between 3 and 6 months post-transplantation, we observed islet-like structures within the grafts (Figure 4 and data not shown) that contained NKX6-1+, INS+, GCG+, SST+, and PP+ monohormonal cells. Trypsin+ and CK19+ cells were also detected at this time point, suggesting that acinar and ductal lineage cells were present in the graft. Interestingly, at this time point, fewer endocrine cells were found within the CK19+ structures.

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