Fig 2: (A) Immunohistochemistry of ZIP4 at days 0, 7, 17, and 21. Representative results from three independent experiments are shown. (B) Staining intensities in images were measured by ImageJ. (C) Immunohistochemistry of ZnT8 at days 0, 7, 17, and 21. Representative results from three independent experiments are shown. (D) Staining intensities in images were measured by ImageJ. Three random fields were analysed. R: RCP petaloid µ-piece, *P < 0.05, **P < 0.01, Fisher’s least significant difference. Error bars represent the standard deviation.

Fig 3: Morphology of the generated NOD-IPCs. (a–d) Immunofluorescence staining of insulin (a), C-peptide (b), PDX-1 (c), and NKX6.1 (d) in NOD-IPCs on day 28. Green: insulin, C-peptide, PDX-1, or NKX6.1; blue: DAPI. Scale bar, 200 µm; scale bar of the larger image, 25 µm. (e–g) Immunofluorescence staining of autoantigens (ICA, GAD65, and ZnT8) in NOD-IPCs on day 28. ICA and ZnT8 were highly expressed, whereas GAD65 was not expressed. Green: ICA, ZnT8, or GAD65; blue: DAPI. Scale bar, 200 µm; scale bar of the larger image, 25 µm. (h) Immunofluorescence staining of PD-L1, which was highly expressed in NOD-IPCs. Green: PD-L1; blue: DAPI. Scale bar, 200 µm; scale bar of the larger image, 25 µm. Islets of NOD mice were used as a control.

Fig 4: Pathological examination of the transplanted NOD-IPCs. (a) H&E-stained transplanted NOD-IPCs were examined under the kidney capsule. RCP µ-pieces were identified at the indicated time points (yellow arrowheads). Upper panels. Original magnification, × 100. Scale bar, 200 µm. Lower panels. Original magnification, × 400. Scale bar, 50 µm. (b–d) Immunohistochemical staining of the NOD-IPCs. These cells showed substantial staining by the anti-insulin and anti-PDX-1 antibodies on Day 30; however, they were barely stained on Day 60. The expression of PD-L1 was maintained in the transplanted NOD-IPCs on Day 30; however, the expression was significantly lower on Day 60. Original magnification, × 400. Scale bar, 50 µm. (e, f) On Day 30, small numbers of CD4+ and CD8+ T cells were observed around the transplanted NOD-IPCs. However, many CD4+ and CD8+ T cells had accumulated around the transplanted NOD-IPCs and infiltrated deeply into the NOD-IPCs clusters by Day 60. The numbers of CD4+ and CD8+ T cells were significantly higher on Day 60 than on Day 30. Original magnification, × 400. Scale bar, 25 µm. The lines in the boxes show the median values (*P < 0.05, Mann–Whitney U test). (g–i) The expression of ICA and ZnT8, which were expressed before transplantation, was maintained after transplantation (no difference in staining intensity between the time points). Conversely, GAD65, which was not expressed in vitro, was expressed in NOD-IPCs after transplantation. Moreover, the staining intensity was significantly higher on Day 60 than on Day 30. Original magnification, × 400. Scale bar, 50 µm. The lines in the boxes show the median values (*P < 0.05, Mann–Whitney U test).