A representative image of hNSE staining in the striatum.

<p>Human graft cells were densely confined to the striatum (arrow) with small amounts of hNSE+fibers that extended dorsally and ventrally from the striatum (arrowheads). Scale bar = 0.5 mm [Iv; lateral ventricle, Str: striatum].</p

Experimental Treatment Conditions.

<p>Treatment Summary Table. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-t001" target="_blank">Table 1</a> summarizes the 4 treatment groups of randomly assigned stroke animals.</p

NSI-566RSC cell grafts attenuate stroke-induced neurologic impairments.

<p>Neurological function was assessed using a battery of neurological tests (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-g002" target="_blank">Figure 2</a>). All animals displayed normal neurological function at Baseline (i.e., prior to stroke). At day 7 post-stroke, all animals exhibited significant impairment in neurological function, indicating that all animals received successful stroke. At 14 days post-stroke onwards, dose-dependent and timing-dependent effects of the treatment were recognized, in that the improvement in neurological performance was in the order of high dose to zero dose as follows: 20,000 cells/µl (D) >10,000 cells/µl (C) >5,000 cells/µl (B)>vehicle infusion only (A). In addition, over time there was a trend of better improvement, with the most significant improvement seen at 56 days post-stroke. <b>*</b>significant <0.05 vs. other treatment groups within time point; <b>^#</b>significant <0.05 vs. other time points.</p

Migration of hNSE+fibers toward the contralateral side of non-infarct region.

<p>Needle tract with hNSE+stain was found in the cortex. Additionally, small amounts of hNSE+fibers crossed the <i>cc contralaterally (inset).</i> Scale bar = 1.0 mm [Iv; lateral ventricle, Str: striatum, ctx: cortex, <i>cc</i>: corpus callosum].</p

NSI-566RSC cell grafts ameliorate stroke-induced motor deficits.

<p>Motor performance was measured by EBST (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-g001" target="_blank">Figure 1</a>). All animals displayed normal motor behavior at Baseline (i.e., prior to stroke). At day 7 post-stroke, all animals exhibited 100% biased swing behaviors, indicating that all animals received successful stroke. At 14 days post-stroke onwards, dose-dependent and timing-dependent effects of the treatment were recognized, in that the improvement in behavioral performance was in the order of high dose to zero dose as follows: 20,000 cells/µl (D) >10,000 cells/µl (C) >5,000 cells/µl (B)>vehicle infusion only (A). In addition, over time there was a trend of better improvement, with the most significant improvement seen at 56 days post-stroke. *significant <0.05 vs. other treatment groups within time point; ^#significant <0.05 vs. other time points.</p

A typical infarct lesion in the striatum and cortex.

<p>The tissue damage covers the cortex and infarct striatum (dash area) and areas lateral to infarct core lesion (not shown here). Note the shrinkage and dissolution of the cells in the penumbra (box) compared to the corresponding contralateral side of the brain (Insets a and b). Scale bar = 1.0 mm [Iv; lateral ventricles, Str: striatum].</p

A representative image of HuNu staining in the striatum.

<p>HuNu+cells were confined to the striatum of rats receiving the lowest dose of graft cells (Group B). Note that small amounts of HuNu staining extended both dorsally and ventrally from the striatum. Scale bar = 0.5 mm [Iv; lateral ventricle, Str: striatum].</p

Behavioral and Histopathological Assessment of Adult Ischemic Rat Brains after Intracerebral Transplantation of NSI-566RSC Cell Lines

<div><p>Stroke is a major cause of death and disability, with very limited treatment option. Cell-based therapies have emerged as potential treatments for stroke. Indeed, studies have shown that transplantation of neural stem cells (NSCs) exerts functional benefits in stroke models. However, graft survival and integration with the host remain pressing concerns with cell-based treatments. The current study set out to investigate those very issues using a human NSC line, NSI-566RSC, in a rat model of ischemic stroke induced by transient occlusion of the middle cerebral artery. Seven days after stroke surgery, those animals that showed significant motor and neurological impairments were randomly assigned to receive NSI-566RSC intracerebral transplants at two sites within the striatum at three different doses: group A (0 cells/µl), group B (5,000 cells/µl), group C (10,000 cells/µl), and group D (20,000 cells/µl). Weekly behavioral tests, starting at seven days and continued up to 8 weeks after transplantation, revealed dose-dependent recovery from both motor and neurological deficits in transplanted stroke animals. Eight weeks after cell transplantation, immunohistochemical investigations via hematoxylin and eosin staining revealed infarct size was similar across all groups. To identify the cell graft, and estimate volume, immunohistochemistry was performed using two human-specific antibodies: one to detect all human nuclei (HuNu), and another to detect human neuron-specific enolase (hNSE). Surviving cell grafts were confirmed in 10/10 animals of group B, 9/10 group C, and 9/10 in group D. hNSE and HuNu staining revealed similar graft volume estimates in transplanted stroke animals. hNSE-immunoreactive fibers were also present within the corpus callosum, coursing in parallel with host tracts, suggesting a propensity to follow established neuroanatomical features. Despite absence of reduction in infarct volume, NSI-566RSC transplantation produced behavioral improvements possibly via robust engraftment and neuronal differentiation, supporting the use of this NSC line for stroke therapy.</p></div

Summary of histological assessment of infarct brains transplanted with NSI-566RSC (n = 10 per group)^*.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-t002" target="_blank">Table 2</a> summarizes the histological assessment of infarct brains that received three different doses of NSI-566RSC cell lines (Group B, C, and D) in comparison with the vehicle (Group A).</p><p>*Quantitative analysis did not find statistical difference in graft size across the three dose groups, either by HuNu or by hNSE stain (<i>p</i> = 0.2002).</p><p>**Freq. of infarct lesion was based on evidence of tissue damage and cellular abnormality such as cell loss, apoptotic bodies in penumbra regions.</p