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

Intra-animal variability in stereological analyses.

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

hFb engraftment does not correlate with behavioral or histological measures of recovery.

<p>A: In contrast to hCNS-SCns, linear regression analysis revealed a positive, but non-significant correlation between hFb engraftment and the number of errors made on the horizontal ladderbeam task <i>(Pearson r = 0.49, p = 0.26, 2-tailed t-test</i>). B–D: Linear regression analyses for the estimated number of hFb and other measures of host recovery revealed no significant correlations between cell engraftment and lesion volume (B) (<i>Pearson r: r = 0.59, p = 0.16, 2-tailed t-test</i>), volume of spared tissue (B) (<i>Pearson r: r = 0.55, p = 0.21, 2-tailed t-test</i>), serotonergic fiber sprouting (C) (<i>Pearson r: r = −0.53, p = 0.22, 2-tailed t-test</i>), NG2 area (D) (<i>Pearson r: r = 0.01, p = 0.98, 2-tailed t-test</i>), and area of the GFAP astroglial scar (D) (<i>Pearson r: r = 0.22, p = 0.63, 2-tailed t-test</i>).</p

Human cell transplantation does not alter angiogenesis.

<p>A: Estimated length of blood vessels at the injury epicenter and at regions 1 mm rostral and 1 mm caudal to the lesion site were quantified using the Space Balls probe of StereoInvestigator. B: The injury epicenter was identified using DAPI immunofluorescent staining within the PE-CAM1-stained sections. C: While there was a trend toward an increase in blood vessel length at regions 1 mm rostral and caudal to the lesion, this difference was not statistically significant (<i>One-way ANOVA: F = 1.87, p = 0.17</i>). D: No significant differences in the blood vessel length were detected between the groups at the injury epicenter (<i>One-way ANOVA: F = 0.65, p = 0.53</i>). Scale Bar = 250 µm for A and B.</p

hCNS-SCns engraftment directly correlates with a quantitative measure of behavior, but not other measures of histological recovery.

<p>A: Linear regression analysis revealed a significant negative correlation between hCNS-SCns engraftment and the number of errors made on the horizontal ladderbeam task (<i>Pearson r = −0.78, p = 0.038, 2-tailed t-test</i>). B-D: Linear regression analyses for the estimated number of hCNS-SCns and other measures of host recovery revealed no significant correlations between cell engraftment and lesion volume (B) (<i>Pearson r = −0.70, p = 0.08, 2-tailed t-test</i>), volume of spared tissue (B) (<i>Pearson r = 0.42, p = 0.34, 2-tailed t-test</i>), serotonergic fiber sprouting (C) (<i>Pearson r = 0.16, p = 0.77, 2-tailed t-test</i>), NG2 area (D) (<i>Pearson r = 0.16, p = 0.77, 2-tailed t-test</i>), and area of the GFAP astroglial scar (D) (<i>Pearson r = −0.69, p = 0.08, 2-tailed t-test</i>). * denotes p<0.05.</p

Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hCNS-SCns.

<p>A: Immunostaining using SC121 revealed cell survival in animals that received hCNS-SCns at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of (A) demonstrated the presence of healthy cells with staining of cytoplasm and processes. C: Animals receiving hCNS-SCns at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the presence of hCNS-SCns with foamy appearance and/or morphological characteristics representative of unhealthy or apoptotic/necrotic cells (arrowheads). E: Quantification for the estimated number of hCNS-SCns 17 weeks post-transplant revealed limited proliferation of hCNS-SCns in animals receiving saline at 16 weeks post-transplant. Dashed line indicates the original transplanted dose of 75,000 hCNS-SCns. DT administration resulted in a significant reduction (80.5%) in the number of hCNS-SCns. <i>** denotes p<0.0001, 1-tailed t-test.</i> Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.</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

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

Number of animals analyzed for individual markers using StereoInvestigator.

<p>Only animals containing three or more sections in which the injury epicenters were visible were included in the analyses.</p