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Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 ± 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats., (Published by Elsevier Inc.)

The goal of the present study was to develop a porcine spinal cord injury (SCI) model, and to describe the neurological outcome and characterize the corresponding quantitative and qualitative histological changes at 4-9 months after injury. Adult Gottingen-Minnesota minipigs were anesthetized and placed in a spine immobilization frame. The exposed T12 spinal segment was compressed in a dorso-ventral direction using a 5-mm-diameter circular bar with a progressively increasing peak force (1.5, 2.0, or 2.5 kg) at a velocity of 3 cm/sec. During recovery, motor and sensory function were periodically monitored. After survival, the animals were perfusion fixed and the extent of local SCI was analyzed by (1) post-mortem MRI analysis of dissected spinal cords, (2) qualitative and quantitative analysis of axonal survival at the epicenter of injury, and (3) defining the presence of local inflammatory changes, astrocytosis, and schwannosis. Following 2.5-kg spinal cord compression the animals demonstrated a near complete loss of motor and sensory function with no recovery over the next 4-9 months. Those that underwent spinal cord compression with 2 kg force developed an incomplete injury with progressive partial neurological recovery characterized by a restricted ability to stand and walk. Animals injured with a spinal compression force of 1.5 kg showed near normal ambulation 10 days after injury. In fully paralyzed animals (2.5 kg), MRI analysis demonstrated a loss of spinal white matter integrity and extensive septal cavitations. A significant correlation between the magnitude of loss of small and medium-sized myelinated axons in the ventral funiculus and neurological deficits was identified. These data, demonstrating stable neurological deficits in severely injured animals, similarities of spinal pathology to humans, and relatively good post-injury tolerance of this strain of minipigs to spinal trauma, suggest that this model can successfully be used to study therapeutic interventions targeting both acute and chronic stages of SCI.