Fig. 2

Transplantation of human iPSC-derived regionalized NPCs. a Representative image of the H&E-stained spinal cord of an SC-type NPC-grafted mouse at 9 weeks after injury. Scale bar, 1000 μm. b Representative immunohistochemical image of STEM121-positive cells in an SC-type graft at 9 weeks after injury. Scale bars, 1000 μm. c Immunohistochemical analysis of transplanted cells to detect regional markers. Regionalized NPCs, which were identified by the human-specific antibody HNA, retained their regional identities after transplantation. Scale bar, 20 μm. d Percentages of regional marker-positive cells among the HNA-positive transplanted cells at 9 weeks after injury (n = 3 mice; mean ± SD; n.d., not detected). e Immunohistochemical analysis of SC-type NPC transplanted cells to detect neuronal, astrocyte, and oligodendrocyte markers. Scale bar, 20 μm. f Percentages of marker-positive cells among the HNA-positive SC-type transplanted cells at 9 weeks after injury (n = 5 mice; mean ± SD). g, h Low (g) and high(h) magnification view of BDA-labeled CST axons with STEM121-positive SC-type transplanted cells. White box indicates the area shown in the high-magnification image. Scale bar, 500 μm (g) and 200 μm (h). i Confocal z-stack images showing synapse formation between CST axons and SC-type grafts. The upper squares show the xz-plane, and the right squares show the yz-plane. Scale bar, 2 μm. j, k Immuno-electron microscopic images of the CST-graft contact region. Immunogold-labeled BDA-positive myelinated CST fibers (J, yellow) and STEM121-positive graft cells (K, purple) were detected. These immunogold-labeled cells frequently formed pre- and post-synaptic structures. Arrowheads, postsynaptic density; scale bar, 1 μm (J, upper), 0.2 μm (J, lower), 1 μm (K, upper), 0.5 μm (K, lower)