Immunohistological characterization in rat spinal cords at the epicenter of injury (asterisks) following transplantation of either (A) heat-killed iPS-OPs or (B) live iPS-OPs, 24 hours after injury.

<p>Human specific anti-nuclei antibody (HNA) staining showed that cells transplanted at the site of injury could survive, differentiate and express myelin basic protein (MBP, red) in iPS-OPs group, compared to the heat-killed iPS-OPs group. (C) Heat-killed iPS-OPs treated spinal cords demonstrated more cavitation and higher glial fibrillary acidic protein (GFAP) (green) expression than (D) spinal cords treated with iPS-OPs. Heat-killed iPS-OPs also expressed less MBP, a mature oligodendrocyte marker, than live iPS-OPs (red). (E) Double immunostaining with CD68 (expressed by macrophages, microglial, and T cells) and MBP shows more CD68+/MBP+ cells in control animals with morphology indicative of active macrophages or microglial engulfment of OPs. (F) Spinal cords injected with iPS-OPs had significantly fewer CD68+/MBP+ cells. Tissues shown here were isolated approximately 2 months after transplantation. DAPI was used to stain rat and human nuclei (blue). Scale bars for A, C, D: 100 μm and B, E, F: 30 μm. (G) Quantitative image analysis shows that more than 70% of HNA+ cells were differentiated MBP+ oligodendrocytes. Only 4.4 ± 1.5% of grafted cells express glial fibrillary acidic protein (GFAP), while no HNA+ cells were detected with co-localized staining for neuronal marker neuron-specific class III beta-tubulin (TUJ1) or NESTIN. Values represent mean ± SEM. * denote statistical significance of human MBP+ oligodendrocytes and MBP- OPs compared to either human-derived neural progenitors (Nestin+), neurons (TUJ1+) or astrocytes (GFAP+) and † denotes statistical significance of human HNA+MBP+ compared to HNA+MBP- OPs by Tukey post hoc tests. Tissue slices encompassing the injury epicenter were stained in intervals for each antibody combination from 4 rats in each treatment group, P < 0.001). (H) Optical density quantification of GFAP staining, which shows significantly higher intensity of GFAP staining in heat-killed iPS-OP compared with iPS-OPs of both BC1 and MR31 lines. (I) Quantification of the number of CD68+ cells shows significantly higher CD68+ cells in heat-killed iPS-OPs, indicative of increased inflammation and macrophage activation, compared with iPS-OPs of both BC1 and MR31 lines. Values in the graphs represent mean ± SEM. * and † denote statistical significance compared to heat-killed iPS-OPs and BC1-OPs, respectively by Tukey post hoc tests (10 identically numbered tissue slices encompassing the injury epicenter from 4 rats were analyzed for each treatment group, P < 0.001).