Extended Data Fig. 2: Reaggregation of dispersed purified human β-cells.
From: Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells
a, Pure β-cells were labelled with GFP and traced in three different culture conditions: in monolayer (‘single β’), β-cell-only aggregation (‘β’), or β-cell aggregation with stromal cells including HUVECs and MSCs (‘β + HM’). Live imaging at indicated days (middle panels) and immunofluorescence at day 7 (right panels) show β-cell-only pseudoislets were self-organized by day 5, whereas β + HM aggregates were constituted in only 1 day. β-cells in β + HM pseudoislets located at the periphery, whereas HM cells formed the core of the aggregates (red and blue, respectively). b, To determine the optimal number of β-cells per pseudoislet, GFP+ transduced β-cells were seeded on aggregation-plate-wells at the indicated densities. After 7 days of culture, aggregates were analysed and found to be uniform in size. Pseudoislet size correlated with the number of cells seeded per well. Human islet cell aggregates with a diameter of 100–150 μm, consisting of 1,000 cells, have been shown to have a comparable function to native islets24; we thus performed reaggregation experiments at 1,000 β-cells per pseudoislet (1,000 β-cells, 129.6 ± 3.1 μm). β-cell aggregates with HM were also analysed. n = 8 pseudoislets for 500 β-cells, n = 8 pseudoislets for 1,000 β-cells, n = 9 pseudoislets for 2000 β-cells, n = 8 pseudoislets for 3,000 β-cells, and n = 52 pseudoislets for 1,000 β-cells + 400 HUVECs + 100 MSCs. c, Immunofluorescence at indicated time points in β-cell pseudoislets and β-cell + HM pseudoislets. d, TUNEL staining (green) showed rare apoptotic cells (0.8%) in β-cell aggregates after 7 days of culture. e, qPCR analyses of INS and PDX1 expression in monolayer and aggregated β-cells showing higher expression of β-cell markers in reaggregated β-cells. Data are expressed as fold change relative to the value in single β-cells. *P = 0.022 in qPCR for INS, *P = 0.026 in qPCR for PDX1, Mann–Whitney test, two-tailed. n = 6 donor samples. f, ELISA measurements of static glucose-stimulated human insulin release at 3 mM (low) and 20 mM (high) glucose showing glucose-responsive C-peptide secretion in both β and β + HM aggregates, but not in single β-cells. *P = 0.012, **P = 0.0037, ****P < 0.0001, two-way repeated-measures ANOVA with Holm–Sidak’s multiple comparisons test, n = 5 for single β-cells, n = 8 for β and β + HM, n = 6 for native islets (all are biological replications from different donors). g, Stimulation index in glucose-stimulated insulin secretion in f exhibiting comparable values among pseudoislets of β and β + HM and native islets. *P = 0.045, **P = 0.0089, *one-way ANOVA with Benjamini, Krieger and Yekutieli’s multiple comparisons test. Images are representative of five (a, c, d) or three (b) independent experiments. ns, not significant. Data are mean ± s.e.m. Scale bars, 25 μm (a), 50 μm (c, d) or 100 μm (b).
