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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(A) Schematic of the protocol used to differentiate hESCs toward pancreatic endoderm. At the final stage of differentiation, cells were treated with NOGGIN, EGF, and nicotinamide, singly or in combinations.(B) Flow-cytometric analysis for GFP was carried out at d13 of differentiation.(C) Average percentage of NKX6-1:GFP+ cells as measured by flow-cytometry analysis at d13 of differentiation in the different treatment groups (−, untreated; N, NOGGIN; E, EGF; NA, nicotinamide; NE, NOGGIN+EGF; NNA, NOGGIN+nicotinamide; NENA, NOGGIN+EGF+nicotinamide). Error bars represent SD of the mean (n = 3 independent experiments).(D) Kinetic analysis of NKX6-1:GFP expression as measured by flow cytometry between d7 and d17 of differentiation. Error bars represent SD from the mean of three experiments. ∗∗p < 0.05, ∗∗∗p < 0.001 using Student’s t test for statistical analysis.(E) qPCR analysis for NKX6-1, PDX1, PTF1A, and SOX9 expression during pancreatic differentiation of the NKX6-1GFP/w hESC line (d7–d17). NKX6-1GFP/w cells were differentiated as described in (A). 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 = 3 independent experiments).See also Figures S1 and S2.
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fig1: NOGGIN, EGF, and Nicotinamide Induce NKX6-1 Expression(A) Schematic of the protocol used to differentiate hESCs toward pancreatic endoderm. At the final stage of differentiation, cells were treated with NOGGIN, EGF, and nicotinamide, singly or in combinations.(B) Flow-cytometric analysis for GFP was carried out at d13 of differentiation.(C) Average percentage of NKX6-1:GFP+ cells as measured by flow-cytometry analysis at d13 of differentiation in the different treatment groups (−, untreated; N, NOGGIN; E, EGF; NA, nicotinamide; NE, NOGGIN+EGF; NNA, NOGGIN+nicotinamide; NENA, NOGGIN+EGF+nicotinamide). Error bars represent SD of the mean (n = 3 independent experiments).(D) Kinetic analysis of NKX6-1:GFP expression as measured by flow cytometry between d7 and d17 of differentiation. Error bars represent SD from the mean of three experiments. ∗∗p < 0.05, ∗∗∗p < 0.001 using Student’s t test for statistical analysis.(E) qPCR analysis for NKX6-1, PDX1, PTF1A, and SOX9 expression during pancreatic differentiation of the NKX6-1GFP/w hESC line (d7–d17). NKX6-1GFP/w cells were differentiated as described in (A). 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 = 3 independent experiments).See also Figures S1 and S2.

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).


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(A) Schematic of the protocol used to differentiate hESCs toward pancreatic endoderm. At the final stage of differentiation, cells were treated with NOGGIN, EGF, and nicotinamide, singly or in combinations.(B) Flow-cytometric analysis for GFP was carried out at d13 of differentiation.(C) Average percentage of NKX6-1:GFP+ cells as measured by flow-cytometry analysis at d13 of differentiation in the different treatment groups (−, untreated; N, NOGGIN; E, EGF; NA, nicotinamide; NE, NOGGIN+EGF; NNA, NOGGIN+nicotinamide; NENA, NOGGIN+EGF+nicotinamide). Error bars represent SD of the mean (n = 3 independent experiments).(D) Kinetic analysis of NKX6-1:GFP expression as measured by flow cytometry between d7 and d17 of differentiation. Error bars represent SD from the mean of three experiments. ∗∗p < 0.05, ∗∗∗p < 0.001 using Student’s t test for statistical analysis.(E) qPCR analysis for NKX6-1, PDX1, PTF1A, and SOX9 expression during pancreatic differentiation of the NKX6-1GFP/w hESC line (d7–d17). NKX6-1GFP/w cells were differentiated as described in (A). 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 = 3 independent experiments).See also Figures S1 and S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig1: NOGGIN, EGF, and Nicotinamide Induce NKX6-1 Expression(A) Schematic of the protocol used to differentiate hESCs toward pancreatic endoderm. At the final stage of differentiation, cells were treated with NOGGIN, EGF, and nicotinamide, singly or in combinations.(B) Flow-cytometric analysis for GFP was carried out at d13 of differentiation.(C) Average percentage of NKX6-1:GFP+ cells as measured by flow-cytometry analysis at d13 of differentiation in the different treatment groups (−, untreated; N, NOGGIN; E, EGF; NA, nicotinamide; NE, NOGGIN+EGF; NNA, NOGGIN+nicotinamide; NENA, NOGGIN+EGF+nicotinamide). Error bars represent SD of the mean (n = 3 independent experiments).(D) Kinetic analysis of NKX6-1:GFP expression as measured by flow cytometry between d7 and d17 of differentiation. Error bars represent SD from the mean of three experiments. ∗∗p < 0.05, ∗∗∗p < 0.001 using Student’s t test for statistical analysis.(E) qPCR analysis for NKX6-1, PDX1, PTF1A, and SOX9 expression during pancreatic differentiation of the NKX6-1GFP/w hESC line (d7–d17). NKX6-1GFP/w cells were differentiated as described in (A). 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 = 3 independent experiments).See also Figures S1 and S2.
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).

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