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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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NOGGIN, EGF and Nicotinamide Induce NKX6-1 Expression in Non-Targeted hESC Lines(A) Flow-cytometric analysis of intracellular NKX6-1. H1 cells and H9 hESCs were differentiated according to a published protocol (Nostro et al., 2011) up to stage 3 and then treated with NOGGIN, EGF and nicotinamide, alone or in combination, for 6 days. Flow-cytometric analysis was performed at d13 of differentiation. Triple treatment was sufficient to induce the highest percentage of NKX6-1+ cells. Error bars represent SD of the mean (n = 3 independent experiments). ∗∗∗p < 0.001 using Student’s t test.(B) qPCR analysis for NKX6-1, PDX1, PTF1A, SOX9, NEUROD1, and NGN3 expression between d7 and d17 of pancreatic differentiation. Expression levels are normalized to the housekeeping gene TBP and compared with adult pancreas (AP, dashed bar). Error bars represent SD of the mean (n = 5 independent experiments).(C) Immunofluorescence image of d13 culture, showing NKX6-1 in green and PDX1 and SOX9 in red. The solid bar is 200 μm.See also Figures S1 and S2.
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fig2: NOGGIN, EGF and Nicotinamide Induce NKX6-1 Expression in Non-Targeted hESC Lines(A) Flow-cytometric analysis of intracellular NKX6-1. H1 cells and H9 hESCs were differentiated according to a published protocol (Nostro et al., 2011) up to stage 3 and then treated with NOGGIN, EGF and nicotinamide, alone or in combination, for 6 days. Flow-cytometric analysis was performed at d13 of differentiation. Triple treatment was sufficient to induce the highest percentage of NKX6-1+ cells. Error bars represent SD of the mean (n = 3 independent experiments). ∗∗∗p < 0.001 using Student’s t test.(B) qPCR analysis for NKX6-1, PDX1, PTF1A, SOX9, NEUROD1, and NGN3 expression between d7 and d17 of pancreatic differentiation. Expression levels are normalized to the housekeeping gene TBP and compared with adult pancreas (AP, dashed bar). Error bars represent SD of the mean (n = 5 independent experiments).(C) Immunofluorescence image of d13 culture, showing NKX6-1 in green and PDX1 and SOX9 in red. The solid bar is 200 μm.See also Figures S1 and S2.

Mentions: To investigate whether this approach for generating NKX6-1+ cells is broadly applicable to other hPSCs, we tested combinations of EGF, NOGGIN, and nicotinamide on H1 and H9 hESC lines, and analyzed the development of NKX6-1+ cells by intracellular flow cytometry. Similar to our observations with the NKX6-1GFP/w line, the addition of NOGGIN, EGF, or nicotinamide alone had little effect, whereas the combination of all three pathway regulators induced populations consisting of up to 60% NKX6-1+ cells (Figure 2A). Titration experiments revealed that the proportion of NKX6-1+ cells increased with increasing concentrations of EGF, up to a maximum of 80% following induction with 100 or 300 ng/ml of the factor (Figure S2A). Additionally, they established optimal concentrations of NOGGIN between 10 and 100 ng/ml and of nicotinamide at 10 mM (Figures S2B and S2C).


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)

NOGGIN, EGF and Nicotinamide Induce NKX6-1 Expression in Non-Targeted hESC Lines(A) Flow-cytometric analysis of intracellular NKX6-1. H1 cells and H9 hESCs were differentiated according to a published protocol (Nostro et al., 2011) up to stage 3 and then treated with NOGGIN, EGF and nicotinamide, alone or in combination, for 6 days. Flow-cytometric analysis was performed at d13 of differentiation. Triple treatment was sufficient to induce the highest percentage of NKX6-1+ cells. Error bars represent SD of the mean (n = 3 independent experiments). ∗∗∗p < 0.001 using Student’s t test.(B) qPCR analysis for NKX6-1, PDX1, PTF1A, SOX9, NEUROD1, and NGN3 expression between d7 and d17 of pancreatic differentiation. Expression levels are normalized to the housekeeping gene TBP and compared with adult pancreas (AP, dashed bar). Error bars represent SD of the mean (n = 5 independent experiments).(C) Immunofluorescence image of d13 culture, showing NKX6-1 in green and PDX1 and SOX9 in red. The solid bar is 200 μm.See also Figures S1 and S2.
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fig2: NOGGIN, EGF and Nicotinamide Induce NKX6-1 Expression in Non-Targeted hESC Lines(A) Flow-cytometric analysis of intracellular NKX6-1. H1 cells and H9 hESCs were differentiated according to a published protocol (Nostro et al., 2011) up to stage 3 and then treated with NOGGIN, EGF and nicotinamide, alone or in combination, for 6 days. Flow-cytometric analysis was performed at d13 of differentiation. Triple treatment was sufficient to induce the highest percentage of NKX6-1+ cells. Error bars represent SD of the mean (n = 3 independent experiments). ∗∗∗p < 0.001 using Student’s t test.(B) qPCR analysis for NKX6-1, PDX1, PTF1A, SOX9, NEUROD1, and NGN3 expression between d7 and d17 of pancreatic differentiation. Expression levels are normalized to the housekeeping gene TBP and compared with adult pancreas (AP, dashed bar). Error bars represent SD of the mean (n = 5 independent experiments).(C) Immunofluorescence image of d13 culture, showing NKX6-1 in green and PDX1 and SOX9 in red. The solid bar is 200 μm.See also Figures S1 and S2.
Mentions: To investigate whether this approach for generating NKX6-1+ cells is broadly applicable to other hPSCs, we tested combinations of EGF, NOGGIN, and nicotinamide on H1 and H9 hESC lines, and analyzed the development of NKX6-1+ cells by intracellular flow cytometry. Similar to our observations with the NKX6-1GFP/w line, the addition of NOGGIN, EGF, or nicotinamide alone had little effect, whereas the combination of all three pathway regulators induced populations consisting of up to 60% NKX6-1+ cells (Figure 2A). Titration experiments revealed that the proportion of NKX6-1+ cells increased with increasing concentrations of EGF, up to a maximum of 80% following induction with 100 or 300 ng/ml of the factor (Figure S2A). Additionally, they established optimal concentrations of NOGGIN between 10 and 100 ng/ml and of nicotinamide at 10 mM (Figures S2B and S2C).

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