Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs

Abstract

Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro.

FIG. 1.

Identification of candidate mesenchymal factors. A: Diagram of the developing pancreas. B: Isolation of different mouse tissues using LCM. Representative images are shown for isolating pancreatic endoderm, pancreatic mesenchyme, and stomach mesenchyme. C: Microarray data show gene expression patterns across different tissues. Yellow indicates genes with high expression levels; blue indicates genes with low expression level. D: List of secreted factors upregulated in pancreatic mesenchyme compared with pancreatic epithelium.

FIG. 2.

Combined treatment with mesenchymal factors affects pancreatic progenitor induction and endocrine differentiation. A: Schematic depicts differentiation procedure and genes used as markers for specific stages of hESC differentiation. ES/iPS, embryonic stem cells or induced pluripotent stem cells; DE, definitive endoderm; PG, primitive gut, PF, posterior foregut; β-cell, pancreatic β-cells. F7–9, cells treated with CF from day 7 to 9; F10–12, cells treated with CF from day 10 to 12. B: Immunostaining of PDX1 and HNF6 on day 9 shows induction of pancreatic progenitors. C: Q-PCR illustrates day 9 PDX1 expression level in samples with or without combined mesenchymal factors (n = 3 independent experiments). The error bars represent the SD for all graphs. Statistical analysis was performed using the Student t test. *P < 0.05. D: Immunostaining of insulin and glucagon shows differentiation of endocrine cells in control and samples treated with combined factors at different time points. E: Q-PCR analysis of day 15 INS expression levels in samples treated with or without combined mesenchymal factors (n = 3 independent experiments). E (inset): Day 15 relative INS expression in F7–9 samples. B: Scale bars = 200 μm. D: Scale bars = 100 μm.

FIG. 3.

Effect of single candidate factors on induction of pancreatic progenitors. A: Single mesenchymal factors tested in combination with R/K/N. Quantification of PDX1 expression in samples treated with single mesenchymal factors compared with control. Control condition is set to “1.” Ten independent fields were used for counting. Statistical analysis was performed using the Student t test. *P < 0.05 and **P < 0.01. B: Percentage of proliferating cells in control and experimental samples treated with single mesenchymal factors. Total number of cells counted for each condition is ≥14,000. C: Percentage of proliferating pancreatic progenitor cells in control and experimental samples treated with single mesenchymal factors. Total number of cells counted for each condition is ≥14,000. D: Immunostaining with PDX1 and pH3 indicates induction of progenitor cells under control, TGF-β2, and TGF-β3 conditions. D: Scale bar = 200 μm in all images.

FIG. 4.

TGF-β signaling is important for efficient induction of pancreatic progenitors but prohibits endocrine differentiation. A: Immunostaining of PDX1 reveals induction of pancreatic progenitor cells in control and TGF-β2/3–treated samples. Each image is a composite of nine individual images at original magnification ×10 (representative ×10 image is shown as white rectangle) to cover a representative area of the culture (higher magnification pictures is marked by dotted lines). B: Quantification of PDX1-positive cells in day 9 samples with and without TGF-β2/3 treatment. Total number of cells counted for each condition is ≥10,000. C: Q-PCR showing day 9 PDX1 expression levels in control and TGF-β2/3-treated samples (n = 2 independent experiments). D: Q-PCR analysis of day 15 PDX1 expression levels in control and TGF-β2/3-treated samples (n = 3 independent experiments). E: Q-PCR analysis of day 15 INS expression levels in control and TGF-β2/3–treated samples (n = 3 independent experiments). F: Immunostaining of PDX1 and insulin in day 15 samples shows induction of endocrine cells in control but not in TGF-β2/3–treated samples. Each image is a composite of 20 individual original magnification ×10 images to cover a representative area of the culture (representative ×10 image is shown as white rectangle). Insets show higher magnification view. B–E: Statistical analysis was performed using the Student t test. *P < 0.05; **P < 0.01. A–F: Scale bars = 400 μm for all images. A and F insets: Scale bars = 200 μm.

FIG. 5.

Short TGF-β2/3 treatment improves endocrine precursor induction as well as endocrine cell differentiation. A: Q-PCR analysis of day 15 INS expression levels in control and experimental samples treated with TGF-β2/3 for different durations (n = 3 independent experiments). B: Immunostaining of PDX1 and HNF6 shows induction of pancreatic progenitor cells in samples treated with TGF-β2/3 for 1 day compared with control. Insets depict higher magnification view. C: Q-PCR analysis of day 12 NGN3 expression levels in control and TGF-β2/3-treated samples (n = 3 independent experiments). D: Immunostaining of insulin and glucagon in day 15 samples indicates induction of endocrine cells in control and experimental samples treated with TGF-β2/3 for 1 day. Statistical analysis in panels A and C was performed using the Student t test. **P < 0.01. B and D: Scale bars = 200 μm for all images. B: Scale bar = 100 μm for all insets. D: Scale bar = 50 μm for all insets.

FIG. 6.

Inhibition of TGF-β pathway further improves endocrine cell differentiation.ic>: Q-PCR analysis is shown for day 12 NGN3 expression levels in control, TGF-β2/3–treated, and TGF-β2/3+ALK5 inhibitor samples (n = 3 independent experiments). B: Q-PCR analysis of day 15 INS expression levels in samples treated with TGF-β2/3 for different lengths, followed by ALK5 inhibitor from day 10 to 15 (n = 3 independent experiments). C: Immunostaining of insulin and glucagon in day 15 samples showing induction of endocrine cells in control and experimental samples treated with ALK5 inhibitor alone (day 10–15) or TGF-β2/3 (day 7) plus ALK5 inhibitor (day 10–15). D: Percentage of insulin-positive cells on day 15 in control samples, samples treated with ALK5 inhibitor alone, and samples treated with TGF-β2/3 plus ALK5 inhibitor (n = 3 independent experiments). Q-PCR analysis of day 15 INS (E) and PDX1 (F) expression levels in control, samples treated with ALK5 inhibitor alone, and with TGF-β2/3 plus ALK5 inhibitor (n = 3 independent experiments). A, B, and D–F: Statistical analysis was performed using the Student t test. *P < 0.05, * *P < 0.01, * * *P < 0.001, and * * * *P < 0.0001. C: Scale bar = 200 μm for all images.

FIG. 7.

Characterization of insulin-producing cells generated from hESCs. Immunostaining with insulin and glucagon (A); C-peptide (B); somatostatin (C); PDX1 (D); NKX2.2 (E); and NKX6.1 (F). D: The arrows mark insulin- and PDX1 double-positive cells; arrowheads mark PDX1-positive, insulin-negative cells. E and F: Arrows mark insulin-producing cells coexpressing NKX2.2 or NKX6.1, respectively. Insets show higher magnification view. G: Q-PCR analysis comparing INS, PDX1, and NKX6.1 expression levels between hESC-derived cultures and adult human islets. H: Corrected C-peptide content in control and samples treated with TGF-β2/3 for 1 day plus ALK5 inhibitor from day 10 to 15. Total C-peptide content is divided by the percentage of insulin-positive cells in culture. I: C-peptide release assay demonstrates cells respond efficiently to KCL stimulation but not to high glucose. Statistical analysis in panels G and H was performed using the Student t test. ***P < 0.001. C–F: Scale bar = 100 μm. C–F (insets): Scale bar = 25 μm. A and B: Scale bar = 200 μm. A and B (insets): Scale bar = 50 μm.