hCNS-SCns differentiation/fate quantification 16 weeks post-transplantation.

<p>Bar graph revealing quantification of hCNS-SCns that expressed the proliferative marker Ki67, the immature neural marker nestin, immature oligodendrocyte marker Olig2, the mature oligodendrocyte marker APC-CC1, the neuronal marker ß-tubulin III and the astrocytic marker GFAP expressed as percentages.</p

Intra-animal variability in stereological analyses.

<p>Intra-animal variability in stereological analyses.</p

Human cytoplasm co-localizes with paranodal protein CASPR.

<p>(<b>A</b>) Orthogonal view of a confocal image of SC121 (red), CASPR (green) and DAPI counterstain (blue). The crosshair reveals co-localization of CASPR with SC121 and orthogonal projection. Arrows indicate additional SC121-positive axons exhibiting compact CASPR-positive paranodes. Arrowheads indicate diffusely distributed CASPR. (<b>B–E</b>) High-power images revealing examples of CASPR and SC121 co-localization. (<b>B</b>) High-power view of area in crosshair from (<b>A</b>). The two discrete CASPR-positive areas are ∼4 µm apart suggesting they are two paranodal regions of a single node. (<b>C</b>) High-power view of another co-localized axon revealing two discrete paranodal regions of a single node. (<b>D, E</b>) Additional high-power examples of SC121 co-localized with CASPR. Left scale bar  = 20 µm, right scale bars  = 1 µm.</p

hCNS-SCns transplantation does not alter lesion volume, spared tissue volume, or glial scar area.

<p>(<b>A</b>) Representative spinal cord stained for GFAP to stereologically quantify lesion volume, indicated by the blue outline, spared tissue volume, quantified 500 µm rostrally and caudally from the lesion edges excluding the lesion, as depicted by the gray box, and the area of dense GFAP expression indicative of glial scarring excluding the lesion, outlined in black. (<b>B</b>) Staining of human GFAP (SC123), indicating rare astrocytic differentiation localized primarily near the injury site that did not exacerbate the glial scar. (<b>C</b>) Lesion volumes quantified using unbiased stereological probe Cavalieri Estimator show no significant difference for any of the three groups (p≥0.91 ANOVA). (<b>D</b>) Spared tissue volumes quantified using unbiased stereological probe Cavalieri Estimator show no significant difference for any of the three groups (p≥0.21 ANOVA). (<b>E</b>) Glial scar areas quantified using unbiased stereological probe Cavalieri Estimator show no significant difference for any of the three groups (p≥0.98 ANOVA). Scale bar  = 1000 µm.</p

hCNS-SCns mostly differentiate into oligodendrocytes and neurons, and few astrocytes.

<p>(<b>A–E</b>) Several human nuclei positive cells, green (<b>C</b>), expressed Olig2 marker revealing immature oligodendrocytes, red (<b>B</b>), DAPI counterstain, blue (<b>A</b>). Arrows indicate a double-labeled cell. Merged confocal image, showing hCNS-SCns expression of Olig2 indicating differentiation to oligodendrocytes (<b>D</b>). Orthogonal view of confocal image showing co-localization of Olig2 and SC101 (<b>E</b>). (<b>F–J</b>) Some human nuclei-positive cells, SC101 green (<b>H</b>) also express the mature oligodendrocyte marker APC-CC1, red (<b>G</b>), DAPI counterstain, blue (<b>F</b>) Arrows indicate a double-labeled cell. Merged confocal image reveals some hCNS-SCns express APC-CC1 expression 16 weeks after transplantation. (<b>I</b>). Orthogonal view of confocal image revealing co-localization of APC-CC1 and human nuclei marker (<b>J</b>). (<b>K–O</b>) Human cytoplasm-positive cells SC121, green (<b>M</b>) also exhibit ß-tubulin III expression, red (<b>L</b>). DAPI counterstain, blue (<b>K</b>). Arrows indicate a double-labeled cell. Merged confocal image, revealing hCNS-SCns expression of ß-tubulin III (<b>N</b>). Orthogonal view of confocal image showing co-localization of ß-tubulin III and SC121 (<b>O</b>). (<b>P–T</b>) Few human cytoplasm cells SC121, red (<b>Q</b>) also expressed and the astrocyte marker GFAP, green (<b>R</b>). DAPI counterstain, blue (<b>P</b>) Arrowhead indicates a non-astrocytic human cell. Co-localization was rare indicating few hCNS-SCns exhibited astrocytic differentiation 16 weeks after transplantation (<b>R</b>). Orthogonal view of confocal image revealing co-localization of GFAP and SC121 (<b>S</b>). Scale bars  = 20 µm and 10 µm in the bottom row.</p

hCNS-SCns promote improved locomotor recovery on multiple tests.

<p>(<b>A</b>) BMS locomotor performance is significantly improved in hCNS-SCns treated animals compared to vehicle controls (repeated measures ANOVA (p≤0.0022). A Bonferroni/Dunn post-hoc analysis at week 16 revealed a significant difference between hCNS-SCns and vehicle control (p≤0.02). There were no significant differences between hFbs and either hCNS-SCns or vehicle. (<b>B</b>) Recovery of coordination was significantly increased in hCNS-SCns treated animals compared to vehicle controls using chi square analysis for observed frequency (p≤0.047, Fisher's Exact Test). No statistically significant differences were found comparing hFbs with vehicle or hCNS-SCns transplanted animals. Error bars are not plotted as these bars represent the absolute percentage of animals reaching criteria. (<b>C</b>) CatWalk gait analysis showed that hCNS-SCns treated animals exhibited significantly increased swing speed compared to vehicle treated animals (p≤0.04, ANOVA, Fisher's PLSD). (<b>D</b>) von Frey analysis of mechanical allodynia showed no significant differences between any of the groups (p>0.05 ANOVA).</p