a, Human islets from T2D donors were dissociated into single cells analysed by flow cytometry. Representative FACS plots showing cells labelled with the pan-endocrine marker HIC1-2B4 and non-β-cell endocrine marker HIC3-2D1220. The purity of sorted islet cells was evaluated. Data are mean ± s.d. FACS plots are representative of three T2D donors. b, c, Reprogramming efficiency into insulin production (percentage of insulin+ GFP+ out of GFP+ cells in b) and qPCR analysis of human INS expression (c), 7 days after aggregation of α-cells transduced with the indicated reprogramming factors. PDX1 and MAFA combined (αPM) trigger the highest reprogramming efficiency. *P = 0.031, ****P < 0.0001 versus αGFP control; ##P = 0.0055 versus αPM, one-way ANOVA with Tukey’s multiple comparisons test. n = 3 different T2D donors. d, Representative immunostaining at culture day 7 in αGFP and αPM pseudoislets from three T2D donors. e, Experimental timeline of experiment 5. Sequential transplantation was performed using human α-cells of T2D donors to rescue STZ-diabetes, followed by anti-glucagon receptor (GCGR) antibody treatment for 2 weeks. Graft was removed 1 week after anti-GCGR antibody therapy. The next week, GCGR antibody treatment was stopped. f, Summary of transplantation experiment using consecutive islet preparations from two different T2D donors. First, 2,300 αPM + HM pseudoislets were transplanted under the kidney capsule of a diabetic STZ-treated NSG mouse. Two weeks later, 1,450 additional αPM + HM pseudoislets were generated and engrafted into the same kidney. g, Random-fed blood glucose levels. Before glucagon inhibition, there is a mild amelioration of hyperglyacemia in the mouse bearing two grafts of T2D αPM pseudoislets, but this is less marked than in the mouse that received T2D islets. After GCGR antibody treatment, glycaemia markedly and quickly drops in both engrafted mice. Graft removal quickly leads to hyperglycaemia, even under glucagon signalling inhibition. h, Glucose tolerance tests before the second transplantation (3 weeks after first transplantation), 4 weeks after the second transplantation, and after graft removal (post Nx). There is improved glucose tolerance in diabetic mice transplanted with αPM + HM pseudoislets (red line) compared with untransplanted diabetic controls (black line). i, Circulating human C-peptide after the first transplantation. The data after the second transplantation are also shown in Fig. 2e. j, In vivo stimulation index (of insulin secretion) after a glucose challenge is similar in native T2D islets and T2D αPM pseudoislets. k, Immunofluorescence of engrafted T2D αPM pseudoislets. Insulin-expressing (red) reprogrammed α-cells (GFP+, green) are abundant and do not contain glucagon (blue). l, m, Reprogramming efficiency (l) and percentage of monohormonal insulin-producing cells (m) in αPM pseudoislets from T2D donors before (pre Tx) and after (post Tx) transplantation. **P = 0.0082, ***P = 0.0005, two-tailed paired t-test. n = 3 donors with T2D from first and second grafts and independent cohort. n, Immunofluorescence for PDX1, MAFA and INS in the graft of T2D intact islets (left) or T2D αPM + HM cells (right) 9 weeks after transplantation. Reprogrammed α-cells express insulin (red), PDX1 (green) and MAFA (blue). o, qPCR analyses in αGFP, αPM aggregates in vitro (before transplantation) and αPM+HM pseudoislets in vivo (after transplantation). Transplanted αPM cells express more INS than cells before transplantation, but still maintained ARX expression. Although endogenous expression of human β-cell transcription factors (PDX1, MAFA and NKX6-1) was not changed significantly 7 days after transduction in vitro, their expression in αPM grafts was significantly increased after transplantation. Gene expression levels were normalized to GFP expression. *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA with Holm–Sidak’s multiple comparisons test. n = 3 different T2D donors for αGFP and αPM in vitro. n = 2 different T2D donors for graft of αPM + HM. p, Transmission electron micrographs of a β-cell in an engrafted T2D islet (left) and of two reprogrammed α-cells in engrafted αPM + HM pseudoislets (right). T2D β-cells do not contain abundant insulin granules, as previously reported61. Reprogrammed α-cells contain abundant β-like granules, with the typical crystalized dense core surrounded by a clear halo. q, TUNEL staining (green) showed very rare apoptosis events (less than 1%) in the graft of αPM + HM pseudoislets 9 weeks after transplantation. In g and h, non-grafted STZ mice (n = 4); STZ mice (n = 1) with αPM+HM graft from 2 donors; STZ mice (n = 1) with native islet graft; healthy mice (n = 2). Data are mean ± s.e.m. Scale bars, 25 μm (d, k, n, q) or 500 nm (p). Images are representative of three different T2D donors’ grafts (k, n, q), and two different T2D donors (p). Source Data