a, Schematic of re-aggregation procedure to remove non-endocrine cells. Cells are enzymatically dissociated and re-aggregated during continued suspension culture. Non-endocrine cells fail to adhere and are removed by filtration. b, Schematic of CD49a enrichment procedure to produce SC-β-cell enriched clusters. Dissociated cells are stained with anti-CD49a PE-conjugated antibody, incubated with anti-PE magnetic microbeads and magnetically separated. The enriched cells are re-aggregated in six-well plates on a rocker. c, t-SNE projection of cells sequenced from native and re-aggregated clusters from a single differentiation show a strong depletion of the non-endocrine population. Cells in both panels were differentiated using protocol v8. d, Immunofluorescence staining for C-peptide, GCG and SLC18A1 shows distinct neighbourhoods in re-aggregated clusters (protocol v8). Images shown are maximum intensity projections from z-stacks. Each panel shows separate representative clusters stained for all markers. Scale bars, 100 μm. e, f, Representative flow cytometry analysis of endocrine cell abundance (from protocol v8), before and after re-aggregation. Endocrine cells express CHGA. g, Summary of population composition (as assayed by flow cytometry) in 60 re-aggregated and 41 native independent differentiations, carried out using protocol v8. Re-aggregations were carried out in spinner flasks. P value computed using two-sided Wilcoxon rank-sum test. In g, h box plots, boxes extend from first to third quartiles, whiskers extend from 5th to 95th percentiles, central line indicates median and box notching indicates 95th percentile confidence interval for median. h, Stimulation index (insulin released at 20 mM glucose versus insulin released at 2 mM) of 52 independent protocol v8 differentiations, with paired native versus re-aggregated comparisons. P value computed using two-sided Wilcoxon signed-rank test. i, Complete data for static GSIS assays, performed as in Extended Data Fig. 2, corresponding to stimulation indices shown in Fig. 4d. Circles are individual technical triplicates and bars show mean of those triplicates. j, Dynamic perifusion assay of glucose-responsive insulin secretion of human islets, native SC-β-cell clusters (stage 6, day 22, v8) and matched SC-β islets produced via magnetic sorting for CD49a. Each point is the mean of three technical replicates, and the vertical bar indicates the s.e. across those triplicates. k, Area under the curve comparing the first low-glucose stimulation and the high-glucose stimulation, normalized to equal effective time in each treatment. Source data