NSI-566 is a primary allogeneic human neural stem cell line derived from a single fetal CNS. Here we present 7 years of follow-up data from a double-blind, randomized, sham-surgery controlled Phase 2a clinical study of NSI-566 intracerebral transplantation in chronic ischemic stroke patients with stable hemiparesis for 6-24 months. Method: A total of 23 patients were randomized into 2 groups where one received 7.2x10 7 NSI-566 cells in about 45 cell deposits around the 3D perimeter of MRI-defined stroke cavity in a single surgery (n=12) and the other received sham surgery (n=11) who were offered NSI-566 transplantation 12 months later. Changes in their upper and lower motor functions were assessed by Fugl-Meyer Motor Score (FMMS, normal=100) by independent blinded stroke rehabilitation specialists. Final follow-up of all available patients was carried out at 48-84 months after the NSI-566 transplantation. Results/Conclusions: Altogether 19 patients received a single dose of NSI-566 of whom 18 completed at least 12 months of follow-up. The degree of motor improvement by FMMS highly correlated with the degree of stroke cavity filling by new tissue from the 566 transplantation. Eight 566 patients showed no filling of the cavity in MRI and no significant change in FMMMS scores for the entire duration. Seven patients had the new tissue primarily within the cavity itself with little merging/integrating with the brain tissues surrounding the cavity. These patients gradually gained partial motor function with clinically meaningful 14-23 points of change from baseline several years after the transplantation. The slow motor gain presumably corresponds to the period for the new tissue from transplantation to integrate with the host brain circuitry. The remaining three patients showed almost complete filling of their cavity with new tissue by 12 months and gained 25-51 FMMS points from baseline of motor improvement that reached 76-87% of normal motor function in combined lower and upper limb movements. The upper extremity function improved up to 36 points and reached up to 89% of normal. The lower extremity function improved up to 8 points and reached up to 82% of normal. The motor improvement of patients who received NSI-566 (n=18) were statistically significant by 2-way ANOVA analysis (p<0.001) against the blinded Sham (n=11). The maximum motor improvement gained remained stable at their final follow-up visit, 74-84 months since the cell transplantation.

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.

Glial cells play a critical role in the development and function of the mammalian central nervous system (CNS). Among other roles, these cells provide the myelin sheath needed for the efficient propagation of impulses along nerve fibers, provide trophic support for neuronal cells, and remove toxins and excess neurotransmitters from the interstitial space. Transplantation of glial cells or glial progenitors into the diseased or injured CNS can provide therapeutic benefits. However, generation of therapeutically useful quantities of glia, in particular oligodendrocytes, is technically challenging. Furthermore, generation of glial precursors from sources such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells poses potential safety risks due to the tumorigenic potential of undifferentiated cells. Here we report a method that enables the efficient generation and expansion of glial precursors from tissue-restricted neural stem cells (NSC). NSC-derived glial precursors can be expanded extensively in culture and retain the capacity to differentiate into oligodendrocytes and astrocytes in vitro and in vivo. Upon transplantation into different animal models of demyelination a substantial proportion of these cells become oligodendrocytes with the capacity to myelinate host axons. These results demonstrate that tissue-restricted human neural stem cells can serve as an efficient source for myelinating oligodendrocytes with therapeutic potential.

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury. Stem Cells Translational Medicine 2019

BACKGROUND:NSI-189 phosphate (NSI-189) is a novel neurogenic molecule with pleiotropic properties, including antidepressant, procognitive, synaptoplastic, and neurotrophic activities demonstrated in preclinical studies. Its antidepressant activity is monoamine-independent. NSI-189 was previously tested in patients with recurrent major depressive disorder in an inpatient setting. METHODS:This study involved 220 patients randomized to an NSI-189 40-mg dose, NSI-189 80-mg dose, or placebo daily for 12 weeks. The study utilized the sequential parallel comparison design, in which the drug effect was tested in 2 separate stages of 6 weeks each. Herein, post-hoc analyses of the data are presented. RESULTS:NSI-189's antidepressant effect increased when the participants' initial baseline depression severity was dichotomized along a Montgomery-Åsberg Depression Rating Scale (MADRS) score of 30. The NSI-189 80-mg dose showed significant benefit over placebo when utilizing the MADRS-6 (P = .046) in the subgroup of patients who were moderately depressed (MADRS < 30) but was not significant in patients who were severely depressed (MADRS ≥30). More pronounced procognitive effects were also observed in the moderate subgroup relative to the severe subgroup or the whole study group, in which 11/36 (31%), 5/36 (14%), or 7/36 (19%) of CogScreen variables significantly improved, respectively. CONCLUSIONS:These results suggest that NSI-189 is effective as a safe adjunctive therapy, with most compelling antidepressant and procognitive benefits noted in patients with moderate depression.

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 x 10(7), 2.4 x 10(7), or 7.2 x 10(7) 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

Despite decades of research, pharmacological therapies for spinal cord motor pathologies are limited. Alternatives using macromolecular, viral, or cell-based therapies show early promise. However, introducing these substances into the spinal cord, past the blood–brain barrier, without causing injury is challenging. We describe a technique for intraspinal injection targeting the lumbar ventral horn in rodents. This technique preserves motor performance and has a proven track record of translation into phase 1 and 2 clinical trials in amyotrophic lateral sclerosis (ALS) patients. The procedure, in brief, involves exposure of the thoracolumbar spine and dissection of paraspinous muscles over the target vertebrae. Following laminectomy, the spine is affixed to a stereotactic frame, permitting precise and reproducible injection throughout the lumbar spine. We have used this protocol to inject various stem cell types, primarily human spinal stem cells (HSSCs); however, the injection is adaptable to any candidate therapeutic cell, virus, or macromolecule product. In addition to a detailed procedure, we provide stereotactic coordinates that assist in targeting of the lumbar spine and instructional videos. The protocol takes ~2 h per animal.

INTRODUCTION: Human neural stem cells (hNSCs) transplantation in several experimental brain injury models has established their therapeutic potential. However, such a promising cell replacement strategy has not been studied in experimental acute subdural hematoma according to current literature. Aside from emergent surgical decompression through evacuation of hematoma and rehabilitation, there are no proven effective treatments exist to aid neurological recovery. Therefore, we aimed to test the feasibility of hNSCs transplantation in rat ASDH decompression model for functional recovery using 2 clinically relevant transplantation approaches. METHODS: Athymic rats were randomized into ASDH and control groups respectively (7-10/group). The ASDH rats then underwent craniotomy for hematoma evacuation mimicking emergent decompression. One week following injury, the rats were subjected to 2 different hNSCs transplantation approaches 1) direct stereotaxic injection at proximal motor area 2) Onlay of hNSC embedded collagen matrix based dural graft (DuraGen) over affected cortical surface (mimicking duroplasty). Behavioral testing was carried out to assess motor function recovery following ASDH induction and decompression utilizing rotarod method. The rats were then sacrificed at 4 and 8 wk timepoint for histoimmunochemistry analysis. RESULTS: There were robust engraftment of the hNSCs in ASDH group at 4 and 8 wk following transplantation. The hNSCs extended neuritis-like projections toward deep parenchyma, resembling neural dendrites. In comparison the direct in situ transplantation group had greater engraftment than the dural draft embedding approach. Immunochemistry with douclecortin, NeuN, and GFAP at 8 wk after transplantation showed that the transplanted hNSCs remained as immature neurons and did not differentiate toward to glial cell lines. In regards to motor recovery, the hNSCs transplant group showed steady increased in motor function up to 4 wk after transplantation. The latency to fall from rotarod was significantly higher in the hNSCs transplantation group compared to control from 2 wk onward post transplantation. CONCLUSION: This is the first study demonstrates robust engraftment of hNSCs via 2 different clinically relevant transplantation approaches in ASDH following craniotomy. There were impressive functional recovery demonstrated in the hNSCs group. The results from this study is promising as an additional neurosurgical intervention to treat functional deficit as a result of ASDH in future.

While peripheral neuropathy is the most common complication of long-term diabetes, cognitive deficits associated with encephalopathy and myelopathy also occur. Diabetes is a risk factor for Alzheimer disease (AD) and increases the risk of progression from mild cognitive impairment to AD. The only current recommendation for preventing or slowing the progression of peripheral neuropathy is to maintain close glycemic control, while there is no recommendation for central nervous system disorders. NSI-189 is a new chemical entity that when orally administered promotes neurogenesis in the adult hippocampus, increases hippocampal volume, enhances synaptic plasticity, and reduces cognitive dysfunction. To establish the potential for impact on peripheral neuropathy, we first showed that NSI-189 enhances neurite outgrowth and mitochondrial functions in cultured adult rat primary sensory neurons. Oral delivery of NSI-189 to murine models of type 1 (female) and type 2 (male) diabetes prevented multiple functional and structural indices of small and large fiber peripheral neuropathy, increased hippocampal neurogenesis, synaptic markers and volume, and protected long-term memory. NSI-189 also halted progression of established peripheral and central neuropathy. NSI-189, which is currently in clinical trials for treatment of major depressive disorder, offers the opportunity for the development of a single therapeutic agent against multiple indices of central and peripheral neuropathy.

Human neural stem cells (hNSCs) transplantation in several brain injury models has established their therapeutic potential. However, the feasibility of hNSCs transplantation is still not clear for acute subdural hematoma (ASDH) brain injury that needs external decompression. Thus, the aim of this pilot study was to test feasibility using a rat ASDH decompression model with two clinically relevant transplantation methods. Two different methods, in situ stereotactic injection and hNSC-embedded matrix seating on the brain surface, were attempted. Athymic rats were randomized to uninjured or ASDH groups (F344/NJcl-rnu/rnu, n = 7–10/group). Animals in injury group were subjected to ASDH, and received decompressive craniectomy and 1-week after decompression surgery were transplanted with green fluorescent protein (GFP)-transduced hNSCs using one of two approaches. Histopathological examinations at 4 and 8 weeks showed that the GFP-positive hNSCs survived in injured brain tissue, extended neurite-like projections resembling neural dendrites. The in situ transplantation group had greater engraftment of hNSCs than matrix embedding approach. Immunohistochemistry with doublecortin, NeuN, and GFAP at 8 weeks after transplantation showed that transplanted hNSCs remained as immature neurons and did not differentiate toward to glial cell lines. Motor function was assessed with rotarod, compared to control group (n = 10). The latency to fall from the rotarod in hNSC in situ transplanted rats was significantly higher than in control rats (median, 113 s in hNSC vs. 69 s in control, P = 0.02). This study first demonstrates the robust engraftment of in situ transplanted hNSCs in a clinically-relevant ASDH decompression rat model. Further preclinical studies with longer study duration are warranted to verify the effectiveness of hNSC transplantation in amelioration of TBI induced deficits.

Alzheimer's disease (AD) is the leading cause of dementia in the aging population and is characterized by neurodegeneration and synaptic dysfunction affecting the cortex and the limbic system, along with Ab deposits and neurofibrillary tangles, leading to cognitive deficits. Currently the FDA-approved drugs to treat AD are symptomatic at best and do not affect the underlying mechanisms. NSI-189, a benzylpiperizine-aminopyridine, is an orally active molecule that stimulates neurogenesis, synaptogenesis and increased hippocampal volume in mice. And recently, it has shown significant antidepressant and pro-cognitive effects in patients with major depressive disorder in a Phase 2 clinical trial. Previously, we presented NSI-189's positive effects on cognition in symptomatic 5xFAD model of AD (AAIC 2018). In this study, we tested its pro-cognitive effects on symptomatic TAPP mice. Wild type mice and mice overexpressing human tau and amyloid precursor protein (TAPP) were used. TAPP mouse carries transgenes for human Aβ protein and human P301L tau mutation to model formation of both plaques and tangles. After confirmation of cognitive impairment at 18–19 weeks of age, one group of TAPP mice and one group of control mice began 12 weeks of oral treatment with NSI-189 at 30 mg/kg. Learning and memory assays using the Barnes Maze and Object recognition tests as well as the learning rotarod test were performed after 6 and 12 weeks of treatment. At the end of the study, brains were collected for Western blot analysis and immunochemistry. Twelve weeks of daily treatment with NSI-189 ameliorated the learning abilities of 7month old TAPP mice as assessed by the Barnes maze and the learning rotarod tests. Memory retention after 2 days without exposure to the Barnes maze was significantly improved and long-term (6 weeks) memory was ameliorated by 12 weeks of treatment with NSI-189 in TAPP mice. Short-term memory assessed using the object recognition test was significantly improved in TAPP mice after 12 weeks of treatment with NSI-189. Daily treatment with NSI-189 significantly reversed learning and memory deficits that were established in the TAPP mouse model of AD.

NSI-189 is a novel neurogenic compound independent of monoamine reuptake pathways. This trial evaluated oral NSI-189 as monotherapy in major depressive disorder. To improve signal detection, the sequential-parallel comparison design (SPCD) was chosen. Two hundred and twenty subjects were randomized to NSI-189 40 mg daily, 80 mg daily, or placebo for 12 weeks. The primary outcome measure was the Montogmery Asberg Depression Rating Scale (MADRS). Secondary subject-rated measures included the Symptoms of Depression Questionnaire (SDQ), the Cognitive and Physical Functioning Scale (CPFQ), the patient-rated version of the Quick Inventory of Depressive Symptomatology Scale (QIDS-SR), and subtests from the CogScreen and Cogstate cognitive tests. MADRS score reduction versus placebo did not reach significance for either dose (40 mg pooled mean difference −1.8, p = 0.22, 80 mg pooled mean difference −1.4, p = 0.34, respectively). However, the 40 mg dose showed greater overall reduction in SDQ (pooled mean difference −8.2; Cohen’s d for Stages 1 and 2 = −0.11 and −0.64, p = 0.04), and CPFQ scores (pooled mean difference −1.9; Cohen’s d for Stages 1 and 2 = −0.28 and −0.47, p = 0.03) versus placebo, as well as QIDS-SR scores in Stage 2 of SPCD (−2.5; Cohen’s d Stages 1 and 2 = −0.03 and −0.68, p = 0.04). The 40 mg dose also showed advantages on some objective cognitive measures of the CogScreen (absolute Cohen’s d ranged between 0.12 and 1.12 in favor of NSI-189, p values between 0.002 and 0.048 for those with overall significance), but not the Cogstate test. Both doses were well tolerated. These findings replicate those of phase 1b study, and warrant further exploration of the antidepressant and pro-cognitive effects of NSI-189.

NSI-189 Phosphate, (4-benzylpiperazin-1-yl)-[2-(3-methyl-butylamino)pyridin-3-yl] methanone is a new chemical entity under development for the treatment of MDD, based upon preclinical data demonstrating stimulation of neurogenesis of human hippocampus-derived neural stem cells in vitro and in mouse hippocampus in vivo. Previous studies have examined the tolerability and efficacy of NSI-189 for treating major depressive disorder (MDD). NSI-189 has shown significant potential as a treatment for MDD, with concurrent improvement of a cognition scale in a small double-blind, placebo-controlled study. The current study evaluated its possible application for the treatment of Angelman Syndrome. Incubation of acute hippocampal slices from wild-type mice with NSI-189 resulted in a time- and dose-dependent increase in the magnitude of long-term potentiation (LTP) elicited by theta burst stimulation (TBS). The same protocol enhanced TBS-induced LTP in acute hippocampal slices from AS mice. A short treatment with daily injections of NSI-189 in AS mice reversed impairments in cognitive and motor functions, while it slightly enhanced performance of WT mice. The effects of NSI-189 on synaptic plasticity and cognitive functions were associated with activation of the TrkB and Akt pathways. These results suggest that NSI-189 could represent a potential treatment for AS patients.

NSI-189, a benzylpiperizine-aminopyridine, is a proprietary, orally active, new chemical entity that stimulates neurogenesis, synaptogenesis and increased hippocampal volume in mice. It has shown significant antidepressant and pro-cognitive effects in patients with major depressive disorder in a Phase II clinical trial. It ameliorated cognitive impairment in a rat model of irradiation-induced brain injury. We have recently found that NSI-189 prevented hippocampal volume decrease in diabetic mice. Transgenic 5xFAD mice carry transgenes for mutant human APP and mutant human PS1 to model familial AD with early Ab deposition and neurodegeneration. Fifteen weeks old mice were treated orally for 12 weeks with NSI-189 at 30 mg/kg. After 6 and 12 weeks of treatment, learning and memory tests (Barnes maze and Object recognition tests) were performed along with anxiety test and repeated testing on the rotarod for assessing motor learning. Six weeks of treatment with NSI-189, significantly restored memory retention in the Barnes maze, while learning abilities were significantly ameliorated by 12 weeks of oral administration of NSI-189. Thirteen weeks of treatment with NSI-189 improved short-term memory capacity of 7month old 5xFAD mice beyond the recognition capacity of control mice in the novel object recognition test. Using the repeated rotarod test showed that the motor performance as well as the learning ability of 5xFAD mice receiving daily NSI-189 treatment were significantly improved compared to 5xFAD mice to a level above control mice values. Anxiety had increased over the study period for untreated mice but daily treatment with NSI-189 partially reduced the developing anxiety for both control and AD mice. Daily treatment with NSI-189 improved learning and memory behaviors as well as reduced developing anxiety in a mouse model of AD.