Fig. 14

1 The process of creating 4n complementation mice using iPSCs without the need for integration. Panel (a) shows the method used to generate integration-free iPSCs. Panel (b) displays the morphology and alkaline phosphatase staining of these iPSCs. Panel (c) presents PCR analysis results, indicating the absence of integration of the reprogramming vector in the iPSC lines tested. The reprogramming plasmid serves as a positive control. Panel (d) demonstrates the normal karyotypes of the iPSCs through G-banding chromosomal analysis. Panel (e) exhibits immunofluorescence staining of pluripotent markers (Nanog, Oct3/4, Sox2, and SSEA-1) in iPSCs. Panel (f) displays the results of RT-PCR analysis, indicating successful differentiation of iPSCs into three germ layers. Panel (g) shows the formation of teratomas containing all three embryonic germ layers when iPSCs are injected into severe-combined-immune-deficiency mice. Panel (h) represents iPSC mice generated through 4n complementation. Finally, panel (i) presents the results of SSLP analysis, which distinguishes mice derived from different iPSC lines. 2 The successful transplantation of skins derived from iPSCs, which were well-tolerated by the host and effectively repaired skin wounds. In (a), a schematic diagram demonstrates the transplantation of skin, islets, and hearts derived from iPSC mice onto different locations of recipient mice. T-cell proliferation or interferon (INF)-γ release assays were used to detect primed T cells. (b) showcases the wound repair achieved through the transplantation of iPSC-derived skin. The transplanted iPSC skin, similar to embryonic stem cell (ESm) and genetically identical skin (syngeneic), survived successfully for over 100 days in recipient mice. Allogeneic skin transplants, serving as negative controls, were rejected within three weeks. Representative images in (b) depict the grafts 20 weeks after transplantation. (c) provides a summary of the survival rates of explanted iPSC skin 20 weeks post-transplantation. ESm and syngeneic skin transplants are shown as positive controls, while allogeneic skin transplants are negative controls. (d) displays histological staining (H&E staining) of iPSC skin isolated from recipient mice eight weeks after transplantation. Allografts were stained one week after transplantation and served as a negative control. iPSC skin explants exhibited normal structures similar to ESm and syngeneic mice, while extensive tissue necrosis was observed in allografts. (e) demonstrates that T-cell infiltration was minimal in iPSC skin explants eight weeks after transplantation. T cells were identified through immunostaining using anti-CD3, anti-CD4, and anti-CD8 antibodies. Sections from the spleen and allogeneic skin grafts (one week after transplantation) were used as positive controls. (f) quantifies the percentage of proliferating cells, while (g) presents an interferon (IFN)-γ release assay to detect primed T cells in recipients of iPSC-derived skin. The quantified results are shown as mean ± s.e.m. of triplicates for each group (syngeneic: n = 3; ESm: n = 6; iPSC: n = 6; allogeneic: n = 3). 3 The effectiveness of iPSC-derived islets in reducing high glucose levels in diabetic mice. In panel (a), the survival of iPSm islets in C57BL/6 hosts is summarized after 8 weeks of transplantation. Panel (b) displays representative images of iPSm islets that were transplanted under kidney capsules, with dot circles indicating the location of the grafted islets. Panel (c) shows the detection of T-cell infiltration in iPSm islets using an anti-CD3 antibody (shown in green). Engrafted islets are labeled with anti-insulin staining (shown in red). In panel (d), the quantification of T-cell proliferation induced by different stimulators is presented, with the mean and standard error of the mean (s.e.m.) shown for each group (syngeneic, ESm, iPSm, and allogeneic). Panel (e) presents the quantification of interferon (IFN)-γ release, again with the mean and s.e.m. shown for each group (syngeneic, ESm, iPSm, and allogeneic). Panel (f) displays the monitoring of blood glucose levels in diabetic mice that were engrafted with allogeneic, syngeneic, and iPSm islets. The different groups are represented by different colors (iPSm in yellow, syngeneic in green, and allogeneic in purple). Finally, in panel (g), the glucose tolerance test conducted 8 weeks after islet transplantation is shown. Diabetic mice engrafted with iPSm islets (represented in green) exhibited efficient response to high-glucose injection similar to mice transplanted with syngeneic islets (represented in purple). 4 Heart transplantation using iPSC-derived cells. (a) Survival rates of mouse hearts derived from iPSCs (iPSm), embryonic stem cells (ESm), syngeneic (genetically identical), and allogeneic (genetically different) transplants in recipient mice. (b) iPSm hearts beat at similar rates to ESm and syngeneic hearts. (c) Transplanted hearts were examined using H&E staining. (d) T-cell infiltration was assessed by staining heart sections with anti-CD3 antibodies (green). No significant T-cell infiltration was observed in iPSm, ESm, and syngeneic mouse hearts from genetically identical recipients, while allografts showed extensive T-cell infiltration (positive controls). Scale bars represent 50 μm. (e) T-cell proliferation and (f) interferon (INF)-γ release were measured to detect activated T cells in mice with iPSm, ESm, syngeneic, and allogeneic heart transplants. (g) Expression of the Zg16 and Hormad1 genes in transplanted skin, islets, and hearts eight weeks after transplantation. Reprinted from [734] with permission from the Springer Nature