Stable Intracerebral Transplantation of Neural Stem Cells for the Treatment of Paralysis Due to Ischemic Stroke.

NSI-566 is a stable, primary adherent neural stem cell line derived from a single human fetal spinal cord and expanded epigenetically with no genetic modification. This cell line is being tested in clinical trials in the U.S. for treatment of amyotrophic lateral sclerosis and spinal cord injury. In a single-site, phase I study, we evaluated the feasibility and safety of NSI-566 transplantation for the treatment of hemiparesis due to chronic motor stroke and determined the maximum tolerated dose for future trials. Three cohorts (n = 3 per cohort) were transplanted with one-time intracerebral injections of 1.2 × 107 , 2.4 × 107 , or 7.2 × 107 cells. Immunosuppression therapy with tacrolimus was maintained for 28 days. All subjects had sustained chronic motor strokes, verified by magnetic resonance imaging (MRI), initiated between 5 and 24 months prior to surgery with modified Rankin Scores [MRSs] of 2, 3, or 4 and Fugl-Meyer Motor Scores of 55 or less. At the 12-month visit, the mean Fugl-Meyer Motor Score (FMMS, total score of 100) for the nine participants showed 16 points of improvement (p = .0078), the mean MRS showed 0.8 points of improvement (p = .031), and the mean National Institutes of Health Stroke Scale showed 3.1 points of improvement (p = .020). For six participants who were followed up for 24 months, these mean changes remained stable. The treatment was well tolerated at all doses. Longitudinal MRI studies showed evidence indicating cavity-filling by new neural tissue formation in all nine patients. Although this was a small, one-arm study of feasibility, the results are encouraging to warrant further studies. Stem Cells Translational Medicine 2019;8:999-1007.

Amelioration of Penetrating Ballistic-Like Brain Injury Induced Cognitive Deficits after Neuronal Differentiation of Transplanted Human Neural Stem Cells.

Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies with penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread perilesional neurodegeneration, similar to that seen in humans following gunshot wound to the head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Toward this objective, green fluorescent protein (GFP) labeled hNSC (400,000 per animal) were transplanted in immunosuppressed Sprague-Dawley (SD), Fisher, and athymic (ATN) PBBI rats 1 week after injury. Tacrolimus (3 mg/kg 2 days prior to transplantation, then 1 mg/kg/day), methylprednisolone (10 mg/kg on the day of transplant, 1 mg/kg/week thereafter), and mycophenolate mofetil (30 mg/kg/day) for 7 days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8 weeks post-transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16 weeks post-transplantation, neither cell proliferation nor glial lineage markers were detected. Transplanted cell morphology was similar to that of neighboring host neurons, and there was relatively little migration of cells from the peritransplant site. By 16 weeks, GFP-positive processes extended both rostrocaudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing the internal capsule, and extending ∼13 mm caudally from transplantation site reaching into the brainstem. In a Morris water maze test at 8 weeks post-transplantation, animals with transplants had shorter latency to platform than vehicle-treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits of durable engraftment and neuronal differentiation. Therefore, these results justify further studies to progress towards clinical translation of hNSC therapy for PTBI.

NSI-189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats.

Enhancing neurogenesis may be a powerful stroke therapy. Here, we tested in a rat model of ischemic stroke the beneficial effects of NSI-189, an orally active, new molecular entity (mol. wt. 366) with enhanced neurogenic activity, and indicated as an anti-depressant drug in a clinical trial (Fava et al., , Molecular Psychiatry, DOI: 10.1038/mp.2015.178) and being tested in a Phase 2 efficacy trial (ClinicalTrials.gov, , ClinicalTrials.gov Identifier: NCT02695472) for treatment of major depression. Oral administration of NSI-189 in adult Sprague-Dawley rats starting at 6 hr after middle cerebral artery occlusion, and daily thereafter over the next 12 weeks resulted in significant amelioration of stroke-induced motor and neurological deficits, which was maintained up to 24 weeks post-stroke. Histopathological assessment of stroke brains from NSI-189-treated animals revealed significant increments in neurite outgrowth as evidenced by MAP2 immunoreactivity that was prominently detected in the hippocampus and partially in the cortex. These results suggest NSI-189 actively stimulated remodeling of the stroke brain. Parallel in vitro studies further probed this remodeling process and demonstrated that oxygen glucose deprivation and reperfusion (OGD/R) initiated typical cell death processes, which were reversed by NSI-189 treatment characterized by significant attenuation of OGD/R-mediated hippocampal cell death and increased Ki67 and MAP2 expression, coupled with upregulation of neurogenic factors such as BDNF and SCF. These findings support the use of oral NSI-189 as a therapeutic agent well beyond the initial 6-hr time window to accelerate and enhance the overall functional improvement in the initial 6 months post stroke.

Autocrine production of IGF-I increases stem cell-mediated neuroprotection.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron (MN) loss. There are currently no effective therapies; however, cellular therapies using neural progenitor cells protect MNs and attenuate disease progression in G93A-SOD1 ALS rats. Recently, we completed a phase I clinical trial examining intraspinal human spinal stem cell (HSSC) transplantation in ALS patients which demonstrated our approach was safe and feasible, supporting the phase II trial currently in progress. In parallel, efforts focused on understanding the mechanisms underlying the preclinical benefit of HSSCs in vitro and in animal models of ALS led us to investigate how insulin-like growth factor-I (IGF-I) production contributes to cellular therapy neuroprotection. IGF-I is a potent growth factor with proven efficacy in preclinical ALS studies, and we contend that autocrine IGF-I production may enhance the salutary effects of HSSCs. By comparing the biological properties of HSSCs to HSSCs expressing sixfold higher levels of IGF-I, we demonstrate that IGF-I production augments the production of glial-derived neurotrophic factor and accelerates neurite outgrowth without adversely affecting HSSC proliferation or terminal differentiation. Furthermore, we demonstrate that increased IGF-I induces more potent MN protection from excitotoxicity via both indirect and direct mechanisms, as demonstrated using hanging inserts with primary MNs or by culturing with organotypic spinal cord slices, respectively. These findings support our theory that combining autocrine growth factor production with HSSC transplantation may offer a novel means to achieve additive neuroprotection in ALS.

Behavioral and histopathological assessment of adult ischemic rat brains after intracerebral transplantation of NSI-566RSC cell lines.

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.

Transplantation of human fetal-derived neural stem cells improves cognitive function following cranial irradiation.

Treatment of central nervous system (CNS) malignancies typically involves radiotherapy to forestall tumor growth and recurrence following surgical resection. Despite the many benefits of cranial radiotherapy, survivors often suffer from a wide range of debilitating and progressive cognitive deficits. Thus, while patients afflicted with primary and secondary malignancies of the CNS now experience longer local regional control and progression-free survival, there remains no clinical recourse for the unintended neurocognitive sequelae associated with their cancer treatments. Multiple mechanisms contribute to disrupted cognition following irradiation, including the depletion of radiosensitive populations of stem and progenitor cells in the hippocampus. We have explored the potential of using intrahippocampal transplantation of human stem cells to ameliorate radiation-induced cognitive dysfunction. Past studies demonstrated the capability of cranially transplanted human embryonic (hESCs) and neural (hNSCs) stem cells to functionally restore cognition in rats 1 and 4 months after cranial irradiation. The present study employed an FDA-approved fetal-derived hNSC line capable of large scale-up under good manufacturing practice (GMP). Animals receiving cranial transplantation of these cells 1 month following irradiation showed improved hippocampal spatial memory and contextual fear conditioning performance compared to irradiated, sham surgery controls. Significant newly born (doublecortin positive) neurons and a smaller fraction of glial subtypes were observed within and nearby the transplantation core. Engrafted cells migrated and differentiated into neuronal and glial subtypes throughout the CA1 and CA3 subfields of the host hippocampus. These studies expand our prior findings to demonstrate that transplantation of fetal-derived hNSCs improves cognitive deficits in irradiated animals, as assessed by two separate cognitive tasks.

BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells.

CNS stem cells are best characterized by their ability to self-renew and to generate multiple differentiated derivatives, but the effect of mitogenic signals, such as fibroblast growth factor 2 (FGF2), on the positional identity of these cells is not well understood. Here, we report that bone morphogenetic protein 2 (BMP2) induces telencephalic CNS stem cells to fates characteristic of neural crest and choroid plexus mesenchyme, a cell type of undetermined lineage in rodents. This induction occurs both in dissociated cell culture and cortical explants of embryonic day 14.5 (E14.5) embryos, but only when cells have been exposed to FGF2. Neither EGF nor IGF1 can substitute for FGF2. An early step in this response is activation of beta-catenin, a mediator of Wnt activity. The CNS stem cells first undergo an epithelial-to-mesenchymal transition and subsequently differentiate to smooth-muscle and non-CNS glia cells. Similar responses are seen with stem cells from E14.5 cortex, E18.5 cortex and adult subventricular zone, but with a progressive shift toward gliogenesis that is characteristic of normal development. These data indicate that FGF2 confers competence for dorsalization independently of its mitogenic action. This rapid and efficient induction of dorsal fates may allow identification of positional identity effectors that are co-regulated by FGF2 and BMP2.