Angiopoietins stimulate pancreatic islet development from stem cells

Abstract

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and β cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.

Figure 1

Outline of the stepwise differentiation procedure and key signature marker gene expressions during iPSC islet development. (a) A schematic diagram of a five-stage islet development protocol. (b,c, d) iPSC-derived cells were characterized for their pancreatic marker gene expressions at stage of definitive endoderm (b), pancreatic progenitor (c), and islet organoid (d). The expression levels were normalized to IMR90 cells. C: control; A: Ang2. Human pancreas RNA (hP) and human islet RNA (hI) were used as positive controls. Results were obtained from four independent differentiation experiments and shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

Representative organogenesis of iPSC-derived islet organoids. At the end of differentiation, the islets were immunofluorescently labeled for (a) C-peptide (CP, red) and glucagon (GCG, green), somatostatin (SST, green) and pancreatic polypeptide (PPY, red). (b) NKX6.1 (green) and CP (red), and MAFA (green) and CP (red). Cells were counterstained with DAPI (grey). Scale bars, 50 μm. Human islets (hIslet) served as a positive control. (c) Semi-quantitative analysis of cellularity of the islets. Image analysis was performed using ImageJ software (n = 7–16 images for each condition). Results are shown as mean ± SD. Different letters indicate significant differences between the groups and p-value represented as *p < 0.05; **p < 0.01; and ***p < 0.001 compared to the control group.

Figure 3

Angiopoietins promote the development of islets with elevated glucose-responsive insulin secretion capacity from differentiation of multiple pluripotent stem cell lines. (a) Parallel glucose-stimulated insulin secretion from islets generated in the absence or presence of Ang2 (n = 6). (b) Insulin stimulation index from consecutive three rounds of low (2 mM)–high (20 mM) glucose challenge measured from iPSC-derived islets by sequential glucose-stimulated insulin secretion analysis. Control (n = 12) and Ang2 (n = 14). (c) Stimulation index of insulin secretion from iPSC-derived islets in the presence of Ang2 at 0 (A0), 4 (A4), 20 (A20), 100 (A100) and 200 ng/ml (A200) (n = 4). Groups with different letters (a or b) represent significant differences. (d-f) Insulin stimulation index of islet organoids generated from iPSC line IMR90 (n = 4) (d), iPSC line DF4 (n = 6) (e), and hESC line H9 (n = 3) (f), respectively. (g) Insulin stimulation index of iPSC-derived islets generated in the presence of Ang1 (n = 4). All the results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.00, and NS: not significant.

Figure 4

Angiopoietins facilitate the development of islets with enhanced glucose-responsive cellular Ca²⁺ influx in insulin-secreting cells. (a) A regimen for imaging cellular Ca²⁺ influx. (b) Representative micrographs of the iPSC-derived islets stained with Fluo-4 upon low (2 mM) or high (20 mM) glucose challenge. Scale bars: 100 μm. (c) Imaging analysis of Ca²⁺ dynamics for a subset of cell populations within an islet organoid. (d) Ca²⁺ dynamics behavior depicted as average of the Fluo-4 fluorescence intensity obtained from each experimental group. These values were from a population of cells within the islets (n = 9) and donor islets (n = 4) over the entire course of sequential glucose stimulations. (e) Fluo-4 stained insulin-secreting cells responding to sequential low–high glucose stimulations all three, two, and one times were circled with magenta, blue, and yellow respectively. Red circles denote cells that did not respond to any glucose challenge. Scale bar: 100 μm. (f) Percentage of cells in the control, Ang2, and Ang1 groups, whose Ca²⁺ influx responded to sequential (low–high) glucose changes. Human islets (hIslet) served as a positive control. Red: nonresponding cells; yellow, blue, and magenta: cells’ Ca²⁺ influx responded to glucose change once, twice, and thrice. More than 400 cells were counted in each islet organoid. All of the experiments were performed in triplicate. (g) The cells’ Ca²⁺ influx changes responding to sequential low–high glucose level challenge. Three islets with single cell number n > 400 and human islets with cell number n > 250 were analyzed. No Res, Res Atl 1, Res Atl 2, and Res 3 denote to cells’ Ca²⁺ influx responded to glucose level changes zero time, at least once, at least twice, and all three times, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Ang2 regulates glucose-responsive insulin secretion through dynamic F-actin remodeling. Representative micrographs of F-actin organization in insulin-secreting cells at (a) 2 mM and (b) 20 mM glucose challenge. White arrowheads indicate thick and highly polymerized F-actin. Yellow arrowheads indicate depolymerized F-actin. Scale bars: 50 µm.

Figure 6

Characterization of F-actin patterning and key molecules involved in glucose-responsive insulin exocytosis. (a) Line scanning analysis of individual insulin secreting cells upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (b) F-actin intensity patterns across the individual insulin-secreting cell upon low or high glucose challenge. (c) Average F-actin intensity determined by line scanning of individual insulin-producing cells upon low or high glucose challenge (n = 15–30 cells at each condition). (d) Representative images of individual C-peptide producing cells analyzed in control and Ang2 groups upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (e) Representative curves of the integrated F-actin intensity from the center to edge of a β cell in the control and Ang2 groups. F-actin intensity in all 360 °C directions was characterized along the radius to obtain radial mean intensity. The x-axis represents distance from center to the edge of a β cell. (f) Average F-actin intensity at low and high glucose conditions determined by radial profiling (n = 15–30 cells at each condition). (g) Relative gene expressions of key molecules involved in glucose-responsive insulin exocytosis. iPSC-derived islets generated in the presence or absence of Ang2 stimulation at Stage V were challenged with low (2 mM) or high (20 mM) glucose, followed by RNA extraction and qRT-PCR analysis. The ratio of gene expression at high to low glucose was assessed. Human donor islets (hIslet) served as controls. Results are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, NS: non-significant.

Figure 7

Ang2 induces the generation of endothelial cells and pericytes in iPSC derived islet microenvironment. (a) Representative images of VE-cadherin and C-peptide immunofluorescence co-staining in islet-like organoids. (b) Representative images of NG2 and C-peptide immunofluorescence co-staining in islet-like organoids. (c,d) The ratio of VE-Cadherin⁺ endothelial area/DAPI area and NG2⁺ pericyte area/DAPI area in islet-like organoids. Scale bars: 50 µm. n = 19–35 islet organoids for each condition. Results are shown as mean ± SD. ****p < 0.0001.

Figure 8

Illustration of Ang2 effect on iPSC islet differentiation and iPSC-derived islet microenvironment. (a) Ang2 supports islet differentiation with the generation of β, α, δ, PP-cells and additionally, promotes the generation of endothelial and pericyte cells. (b-c) iPSC-derived islets under Ang2 cue showed Ca²⁺ influx and regulated activity of CDC42-RAC1 pathway synchronous with glucose level change, leading to F-actin remodeling aided by gelsolin for physiologically functional insulin exocytosis. Created with BioRender.com.