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We report the long-term results for a phase 1 study of neural stem cell transplantation for chronic spinal cord injury. The trial was registered on ClinicalTrials.gov as NCT01772810. The primary outcome of the trial was to test the feasibility and safety of human spinal cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury in four subjects with thoracic two to thoracic twelve spinal cord injury. Here, we report that all four subjects tolerated the stem cell implantation procedure well, and two subjects had durable electromyography-quantifiable evidence of neurological improvement as well as increased neurological motor and sensory scores at five years post-transplantation., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of removal of spinal instrumentation, laminectomy, and durotomy, followed by six midline bilateral stereotactic injections of NSI-566 cells. All subjects tolerated the procedure well and there have been no serious adverse events to date (18-27 months post-grafting). In two subjects, one to two levels of neurological improvement were detected using ISNCSCI motor and sensory scores. Our results support the safety of NSI-566 transplantation into the SCI site and early signs of potential efficacy in three of the subjects warrant further exploration of NSI-566 cells in dose escalation studies. Despite these encouraging secondary data, we emphasize that this safety trial lacks statistical power or a control group needed to evaluate functional changes resulting from cell grafting., (Copyright © 2018. Published by Elsevier Inc.)

Introduction: Intraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury., Methods: Three-month-old female Sprague-Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies., Results: Intraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons., Conclusions: Peri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.