Fig. 5: Both PDX1^LOW and PDX1^HIGH β-cells contribute to human islet function.

a, b Ad-M3C increases Neurog3, Pdx1 and MafA expression (a), while no differences are detected in native NEUROG3, PDX1 and MAFA expression (b) (n = 4–8 donors). c Ad-M3C increases the proportion of cells expressing high PDX1 levels (B-hMAT) (inset is the non-normalized B-hNORM distribution fitted with a polynomial) (n = 13 islets/4 donors; two-way ANOVA, Bonferroni’s multiple comparison) (F = 4.14, DF = 20). d Representative images showing loss of PDX1^LOW cells in B-hMAT islets (detected using a PDX1 antibody with reactivity against mouse and human protein) (scale bar = 42.5 µm). e PDX1 and INS1 are positively correlated in individual cells from B-hNORM islets (n = 220 cells). f–h Ca²⁺ traces (f) showing decreased responsiveness to glucose (g) and KCl (h) in B-hMAT islets (n = 16 islets/3 donors; unpaired t-test). i, j as for (f–h), but representative images (scale bar = 25 µm) showing loss of glucose-stimulated Ca²⁺ rises in B-hMAT but not B-hNORM islets (i), despite no differences in the proportion of responsive cells (j) (n = 16 islets/3 donors; unpaired t-test). k The VDCC and Na⁺ channel subunits CACNA1G, CACNA1C, CACNA1D, SCN1B, SCN3A and SCN8A are all downregulated in B-hMAT islets (n = 4–6 donors; paired t-test). l–o GJD2 expression (l) is decreased in B-hMAT islets (n = 6 donors; paired t-test), which is associated with a decrease in the number of hubs (circled in red) (m) and coordinated β-cell-β-cell activity (connectivity) (n and o) (representative traces are from ‘connected’ cells; raster plots show intensity over time) (n = 7–8 islets/3 donors; unpaired t-test). p–r Non-normalized Insulin secretion is similar in B-hMAT and B-hNORM islets (p), although B-hMAT islets only release a fraction of their total insulin (q and r) (% insulin content = secreted insulin / total insulin) (n = 17–18 replicates/5 donors; unpaired t-test and two-way ANOVA, Bonferroni’s multiple comparison). s Schematic showing proposed changes occurring in β-cells in B-hMAT islets. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant. Color scale shows Ca²⁺ as min (0%) to max (100%) value. GCaMP6-genetically-encoded Ca²⁺ indicator; VDCC-voltage-dependent Ca²⁺ channels; VGSC-voltage-gated Na⁺ channels; GJD2-Gap junction delta-2 protein encoding Connexin-36.