Fig 1: Beta cell-specific loss of Mig6 does not protect against STZ-induced diabetesA. Pancreas and beta cell-specific null mice were administered STZ (35 mg/kg in saline i.p.) for 5 consecutive days. A glucose tolerance test was performed on the third and twentieth day following STZ. Isolated islets or whole pancreata were harvested on days 3 and 21 after STZ. B. Blood glucose levels were measured in mice after a glucose bolus of 1.5 g/kg i.p. C. AUC was calculated. D-E. Representative images and quantification of pancreatic Ins+ beta cell area determined by IHC labeling of whole pancreata of CON, PKO, and BKO mice. n = 6-12; ANOVA, p values: * <0.05, ** <0.01, *** <0.00; data reported as means ± SEM.
Fig 2: Inverse correlation between cellular senescence load and proliferative capacity. (A) Overall proliferation quantification of 6–9-month-old INK-ATTAC animals, ncontrol = 14, and ntreated = 13; (B) Percentage of islets with proliferating cells in 6 and 8/9-month-old INK-ATTAC mice measured by the percentage of BrDU+ Ins+ cells, n6month = 15, and n8–9month = 12; (C) Correlation between percentage of non-senescent FLAG- islets (negative for FLAG staining) and proliferation (positive nuclear staining of BrDU) in 6–9-month-old (n = 19) INK-ATTAC mice. Line of best fit is shown along with dotted lines indicating 95% confidence intervals. P-value was calculated using the null hypothesis that the slope of the best-fit line equals 0.
Fig 3: Proof-of-concept examples and conceptual illustration of downstream assays compatible with the multiwell-based static 3D culture system(A–C) Representative images (A), quantification of cell number per well (B), and heatmap (C) showing well-to-well consistency by calculating percentage deviation from plate mean (see method details). n = 60 wells from one representative experiment. Scale bars, 100 µm.(D and E) Heatmap (D) and quantification (E) showing stimulation index of stage 6–7 clusters per well in static GSIS assays. n = 60 wells from one representative experiment. **p < 0.01 versus stage 6 clusters, unpaired two-tailed t test.(F and G) Representative immunostaining images (F) and ratio of GCG+ to INS+ cells (G) from stage 6–7 clusters across wells. n = 70–80 clusters from 60 wells from one representative experiment. **p < 0.01 versus stage 6 clusters, unpaired two-tailed t test. Scale bars, 100 µm.(H and I) Representative images in duplicates (H) and quantification of mean GFP intensity (I) from an example of optical screens for induction of INS expression (indicated by INSGFP) in stage 7 clusters. S7 basal medium (S7b; MCDB131 medium plus ALK5iII, T3, ITS-X, BSA, heparin, and zinc sulfate). Vehicle control, 0.1% DMSO. n = 30–40 clusters per condition from one representative experiment. ns, not significant, *p < 0.05, **p < 0.01 versus S7b, one-way ANOVA with Dunnett test for multiple comparisons. Scale bars, 750 µm.(J) An example of static GSIS screen for improvement of insulin secretion from stage 7-clusters. Vehicle control, 0.1% DMSO. n = 3 wells including 15–25 clusters per condition from one representative experiment. ns, not significant, *p < 0.05, **p < 0.01 versus S7 complete medium (S7c), one-way ANOVA with Dunnett test for multiple comparisons.(K–N) Graphical abstract (K) and examples (L–N) of various downstream assays for analyzing differentiation cultures with the 96-well-based static 3D system.
Fig 4: Pancreatic loss of Mig6 alters endocrine cell fate without impacting functional beta cell massA. Representative immunofluorescence images of pancreas tissue sections from 10-week-old mice show the distribution of beta cell (INS+), alpha cell (GCG+), and delta cell (SST+) positive area. B. Data was quantified as a percent of total islet area. C. Raw area (µm2) of each endocrine cell type, which collectively yielded the total islet size. D. Islet perimeter was independently quantified. E. Islet circularity was calculated as the ratio of the longest to shortest axis. F. Alpha cell number was manually counted. G. Alpha cell size, calculated as the total alpha positive area / # alpha cells per islet cross-section. H-I. Glucagon positive area / pancreas area was determined by IHC labeling of whole pancreata. n = 6-8 mice; ANOVA or 2-tailed student t-test; p values: * <0.05, ** <0.01, *** <0.001; data reported as means ± SEM.
Fig 5: Generation of insulin-secreting hPSC-islets with the static 3D culture method(A–C) Perifusion assays showing insulin secretion from stage 7 hPSC-islets by both methods (A and B, n = 5) and human islets (C, n = 3). G, glucose; Ex-4, exendin-4. Bottom panel, fold changes over 3.3 mM glucose.(D and E) Total insulin (D) and glucagon (E) content of stage 7 hPSC-islets by both methods (n = 6) and human islets (n = 6). Unpaired two-tailed t test (ns, not significant, *p < 0.05) for comparison between shaker and static. One-way ANOVA with Dunnett test for multiple comparisons (**p < 0.01) was used to compare hPSC-islets by both methods with human islets.(F and G) Representative images of stage 7 hPSC-islets by both methods (F) and human islets (G) stained for various islet cell types and markers. EC, enterochromaffin. INS expression was indicated by INSGFP in hPSC-islets derived from Mel1 INSGFP/W hESCs or insulin antibody staining in human islets. Scale bars, 100 µm.(H and I) Quantification of the percentage of indicated cell types in total population (H) and the percentage of indicated factors co-expressed with INS in INS+ cell population (I) of stage 7 hPSC-islets by both methods (n = 4) and human islets (n = 4). Unpaired two-tailed t test (ns, not significant, *p < 0.05, **p < 0.01) for comparison between shaker and static. One-way ANOVA with Dunnett test for multiple comparisons (ns, not significant, *p < 0.05, **p < 0.01) was used to compare hPSC-islets by both methods to human islets.
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