Transplantation of a vascularized pedicle of hemisected spinal cord to establish spinal cord continuity after removal of a segment of the thoracic spinal cord: A proof-of-principle study in dogs.

INTRODUCTION: Glial scar formation impedes nerve regeneration/reinnervation after spinal cord injury (SCI); therefore, removal of scar tissue is essential for SCI treatment. AIMS: To investigate whether removing a spinal cord and transplanting a vascularized pedicle of hemisected spinal cord from the spinal cord caudal to the transection can restore motor function, to aid in the treatment of future clinical spinal cord injuries. We developed a canine model. After removal of a 1-cm segment of the thoracic (T10-T11) spinal cord in eight beagles, a vascularized pedicle of hemisected spinal cord from the first 1.5 cm of the spinal cord caudal to the transection (cut along the posterior median sulcus of the spinal cord) was transplanted to bridge the transected spinal cord in the presence of a fusogen (polyethylene glycol, PEG) in four of the eight dogs. We used various forms of imaging, electron microscopy, and histologic data to determine that after our transplantation of a vascular pedicled hemisection to bridge the transected spinal cord, electrical continuity across the spinal bridge was restored. RESULTS: Motor function was restored following our transplantation, as confirmed by the re-establishment of anatomic continuity along with interfacial axonal sprouting. CONCLUSION: Taken together, our findings suggest that SCI patients-who have previously been thought to have irreversible damage and/or paralysis-may be treated effectively with similar operative techniques to re-establish electrical and functional continuity following SCI.

Reactive oligodendrocyte progenitor cells (re-)myelinate the regenerating zebrafish spinal cord.

Spinal cord injury (SCI) results in loss of neurons, oligodendrocytes and myelin sheaths, all of which are not efficiently restored. The scarcity of oligodendrocytes in the lesion site impairs re-myelination of spared fibres, which leaves axons denuded, impedes signal transduction and contributes to permanent functional deficits. In contrast to mammals, zebrafish can functionally regenerate the spinal cord. Yet, little is known about oligodendroglial lineage biology and re-myelination capacity after SCI in a regeneration-permissive context. Here, we report that, in adult zebrafish, SCI results in axonal, oligodendrocyte and myelin sheath loss. We find that OPCs, the oligodendrocyte progenitor cells, survive the injury, enter a reactive state, proliferate and differentiate into oligodendrocytes. Concomitantly, the oligodendrocyte population is re-established to pre-injury levels within 2 weeks. Transcriptional profiling revealed that reactive OPCs upregulate the expression of several myelination-related genes. Interestingly, global reduction of axonal tracts and partial re-myelination, relative to pre-injury levels, persist at later stages of regeneration, yet are sufficient for functional recovery. Taken together, these findings imply that, in the zebrafish spinal cord, OPCs replace lost oligodendrocytes and, thus, re-establish myelination during regeneration.

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines.

Spinal cord injury (SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we transplanted adult spinal cord tissues (aSCTs) to replace lost tissues and facilitate recovery in a rat SCI model. However, rodents display considerable differences from human patients in the scale, anatomy and functions of spinal cord systems, and responses after injury. Thus, use of a large animal SCI model is required to examine the repair efficiency of potential therapeutic approaches. In this study, we transplanted allogenic aSCTs from adult dogs to the lesion area of canines after complete transection of the thoracic spinal cord, and investigated the long-term cell survival and functional recovery. To enhance repair efficiency, a growth factor cocktail was added during aSCT transplantation, providing a favorable microenvironment. The results showed that transplantation of aSCTs, in particular with the addition of growth factors, significantly improves locomotor function restoration and increases the number of neurofilament-, microtubule-associated protein 2-, 5-hydroxytryptamine-, choline acetyltransferase- and tyrosine hydroxylase-positive neurons in the lesion area at 6 months post-surgery. In addition, we demonstrated that donor neurons in aSCTs can survive for a long period after transplantation. This study showed for the first time that transplanting aSCTs combined with growth factor supplementation facilitates reconstruction of injured spinal cords, and consequently promotes long lasting motor function recovery in a large animal complete transected SCI model, and therefore could be considered as a possible therapeutic strategy in humans.

Intracerebroventricular Delivery of Human Umbilical Cord Mesenchymal Stem Cells as a Promising Therapy for Repairing the Spinal Cord Injury Induced by Kainic Acid.

Spinal cord injury (SCI) is a common pathological condition that leads to permanent or temporal loss of motor and autonomic functions. Kainic acid (KA), an agonist of kainate receptors, a type of ionotropic glutamate receptor, is widely used to induce experimental neurodegeneration models of CNS. Mesenchymal Stem Cells (MSC) therapy applied at the injured nervous tissue have emerged as a promising therapeutic treatment. Here we used a validated SCI experimental model in which an intraparenchymal injection of KA into the C5 segment of rat spinal cord induced an excitotoxic lesion. Three days later, experimental animals were treated with an intracerebroventricular injection of human umbilical cord (hUC) MSC whereas control group only received saline solution. Sensory and motor skills as well as neuronal and glial reaction of both groups were recorded. Differences in motor behavior, neuronal counting and glial responses were observed between hUC-MSC-treated and untreated rats. According to the obtained results, we suggest that hUC-MSC therapy delivered into the fourth ventricle using the intracerebroventricular via can exert a neuroprotective or neurorestorative effect on KA-injected animals.

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.

Transplantation of adult spinal cord grafts into spinal cord transected rats improves their locomotor function.

Grafted embryonic central neural tissue pieces can recover function of hemisected spinal cord in neonatal rats and promote axonal growth in adults. However, spinal cord segments from adults have not been used as donor segments for allogeneic transplantation. Here, we utilized adult spinal cord tissue grafts (aSCGs) as donor constructs for repairing complete spinal cord injury (SCI). Moreover, to provide a favourable microenvironment for SCI treatment, a growth factor cocktail containing three growth factors (brain-derived neurotrophic factor, neurotrophin-3 and vascular endothelial growth factor), was applied to the aSCG transplants. We found that the locomotor function was significantly improved 12 weeks after transplantation of aSCGs into the spinal cord lesion site in adult rats. Transplantation of aSCGs combined with these growth factors enhanced neuron and oligodendrocyte survival and functional restoration. These encouraging results indicate that treatment of complete SCI by transplanting aSCGs, especially in the presence of growth factors, has a positive effect on motor functional recovery, and therefore could be considered as a possible therapeutic strategy for SCI.

Minimally Invasive and Regenerative Therapeutics.

Advances in biomaterial synthesis and fabrication, stem cell biology, bioimaging, microsurgery procedures, and microscale technologies have made minimally invasive therapeutics a viable tool in regenerative medicine. Therapeutics, herein defined as cells, biomaterials, biomolecules, and their combinations, can be delivered in a minimally invasive way to regenerate different tissues in the body, such as bone, cartilage, pancreas, cardiac, skeletal muscle, liver, skin, and neural tissues. Sophisticated methods of tracking, sensing, and stimulation of therapeutics in vivo using nano-biomaterials and soft bioelectronic devices provide great opportunities to further develop minimally invasive and regenerative therapeutics (MIRET). In general, minimally invasive delivery methods offer high yield with low risk of complications and reduced costs compared to conventional delivery methods. Here, minimally invasive approaches for delivering regenerative therapeutics into the body are reviewed. The use of MIRET to treat different tissues and organs is described. Although some clinical trials have been performed using MIRET, it is hoped that such therapeutics find wider applications to treat patients. Finally, some future perspective and challenges for this emerging field are highlighted.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

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.

Deriving Dorsal Spinal Sensory Interneurons from Human Pluripotent Stem Cells.

Cellular replacement therapies for neurological conditions use human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived neurons to replace damaged or diseased populations of neurons. For the spinal cord, significant progress has been made generating the in-vitro-derived motor neurons required to restore coordinated movement. However, there is as yet no protocol to generate in-vitro-derived sensory interneurons (INs), which permit perception of the environment. Here, we report on the development of a directed differentiation protocol to derive sensory INs for both hESCs and hiPSCs. Two developmentally relevant factors, retinoic acid in combination with bone morphogenetic protein 4, can be used to generate three classes of sensory INs: the proprioceptive dI1s, the dI2s, and mechanosensory dI3s. Critical to this protocol is the competence state of the neural progenitors, which changes over time. This protocol will facilitate developing cellular replacement therapies to reestablish sensory connections in injured patients.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

STUDY DESIGN: Systematic review. OBJECTIVE: The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. SUMMARY OF BACKGROUND DATA: Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. METHODS: The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. RESULTS: Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. CONCLUSION: Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. LEVEL OF EVIDENCE: 5.

Modified acellular nerve-delivering PMSCs improve functional recovery in rats after complete spinal cord transection.

Due to the poor regeneration capacity of neurons and the inhibitory microenvironment, spontaneous regeneration in spinal cord injury (SCI) remains challenging. Tissue engineering is considered a promising approach for enhancing the regeneration of SCI by reconstructing the inherent structure and improving the microenvironment. In this study, the possibility of engineering a nerve complex, which is constructed by acellular nerve delivering placenta mesenchymal stem cells (PMSCs), was assessed for the recovery of a transected spinal cord. Modified acellular nerve grafts were developed, and PMSCs labeled with green fluorescent protein (GFP) were seeded on the graft to construct the engineered nerve complex. Then, the engineered nerve complex was implanted into a 2 mm-length transected gap of the spinal cord. Four weeks after the transplantation, numerous surviving PMSCs were observed in the lesion cavity by immunofluorescence staining. Moreover, co-localization between GFP and neurofilament-200 (NF200) and Neuronal Class III ß-Tubulin (Tuj1) was observed at the bridge interface. The PMSCs-graft group exhibited significant function improvement as evaluated by the Basso, Beattie and Bresnahan (BBB) locomotion score and footprint analysis. Eight weeks after surgery, the evoked response was restored in the PMSCs-graft group and numerous thick myelin sheathes were observed compared to that in the control groups. Collectively, our findings suggest that the nerve complex prepared by acellular nerve delivering PMSCs enhanced the structure and function regeneration of the spinal cord after SCI.

The Therapeutic Effectiveness of Delayed Fetal Spinal Cord Tissue Transplantation on Respiratory Function Following Mid-Cervical Spinal Cord Injury.

Respiratory impairment due to damage of the spinal respiratory motoneurons and interruption of the descending drives from brainstem premotor neurons to spinal respiratory motoneurons is the leading cause of morbidity and mortality following cervical spinal cord injury. The present study was designed to evaluate the therapeutic effectiveness of delayed transplantation of fetal spinal cord (FSC) tissue on respiratory function in rats with mid-cervical spinal cord injury. Embryonic day-14 rat FSC tissue was transplanted into a C4 spinal cord hemilesion cavity in adult male rats at 1 week postinjury. The histological results showed that FSC-derived grafts can survive, fill the lesion cavity, and differentiate into neurons and astrocytes at 8 weeks post-transplantation. Some FSC-derived graft neurons exhibited specific neurochemical markers of neurotransmitter (e.g., serotonin, noradrenalin, or acetylcholine). Moreover, a robust expression of glutamatergic and γ-aminobutyric acid-ergic fibers was observed within FSC-derived grafts. Retrograde tracing results indicated that there was a connection between FSC-derived grafts and host phrenic nucleus. Neurophysiological recording of the phrenic nerve demonstrated that phrenic burst amplitude ipsilateral to the lesion was significantly greater in injured animals that received FSC transplantation than in those that received buffer transplantation under high respiratory drives. These results suggest that delayed FSC transplantation may have the potential to repair the injured spinal cord and promote respiratory functional recovery after mid-cervical spinal cord injury.

3D meshes of carbon nanotubes guide functional reconnection of segregated spinal explants.

In modern neuroscience, significant progress in developing structural scaffolds integrated with the brain is provided by the increasing use of nanomaterials. We show that a multiwalled carbon nanotube self-standing framework, consisting of a three-dimensional (3D) mesh of interconnected, conductive, pure carbon nanotubes, can guide the formation of neural webs in vitro where the spontaneous regrowth of neurite bundles is molded into a dense random net. This morphology of the fiber regrowth shaped by the 3D structure supports the successful reconnection of segregated spinal cord segments. We further observed in vivo the adaptability of these 3D devices in a healthy physiological environment. Our study shows that 3D artificial scaffolds may drive local rewiring in vitro and hold great potential for the development of future in vivo interfaces.

Human neural progenitors differentiate into astrocytes and protect motor neurons in aging rats.

Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population.

Transplantation of olfactory ensheathing cells promotes partial recovery in rats with experimental autoimmune encephalomyelitis.

OBJECTIVE: This study was to investigate the efficacy of olfactory ensheathing cell (OEC) transplantation on experimental autoimmune encephalomyelitis (EAE). METHODS: EAE models were established by guinea pig spinal cord homogenate (GPSCH) immunization in Lewis rats. OECs were purified and cultured from the olfactory nerve layer of SD rats, and then transplanted to the EAE models through the vena caudalis (Group A) or into the lateral cerebral ventricle (Group B). Neurological function scores and body weights were daily recorded following transplantation, and histological analysis was performed to assess the pathological changes in EAE rats. RESULTS: Cultured cells mainly exhibited bipolar or tripolar morphology, and the majority of these cells were positive for NGFR p75 staining. Neurological function scoring and the body weight measurement showed that, OEC transplantation could significantly improve the performance of EAE rats, and similar results were observed for the transplantation through the vena caudalis and into the lateral cerebral ventricle. Moreover, the transplanted OECs accumulated to the lesions in the brains of EAE rats, in spite of the different transplantation approaches. However, no significant differences in histopathology (HE and LFB staining) were observed between the OEC-transplanted groups and the control group. CONCLUSION: OEC transplantation could exert beneficial effects in the treatment of EAE, no matter which the cells were transplanted through the vena caudalis or into the lateral cerebral ventricle. Our findings might provide evidence for the clinical treatment of multiple sclerosis with cell transplantation.

Neuroprotection and immunomodulation by xenografted human mesenchymal stem cells following spinal cord ventral root avulsion.

The present study investigates the effects of xenotransplantation of Adipose Tissue Mesenchymal Stem Cells (AT-MSCs) in animals after ventral root avulsion. AT-MSC has similar characteristics to bone marrow mesenchymal stem cells (BM-MSCs), such as immunomodulatory properties and expression of neurotrophic factors. In this study, Lewis rats were submitted to surgery for unilateral avulsion of the lumbar ventral roots and received 5 × 10(5) AT-MSCs via the lateral funiculus. Two weeks after cell administration, the animals were sacrificed and the moto neurons, T lymphocytes and cell defense nervous system were analyzed. An increased neuronal survival and partial preservation of synaptophysin-positive nerve terminals, related to GDNF and BDNF expression of AT-MSCs, and reduction of pro-inflammatory reaction were observed. In conclusion, AT-MSCs prevent second phase neuronal injury, since they suppressed lymphocyte, astroglia and microglia effects, which finally contributed to rat motor-neuron survival and synaptic stability of the lesioned motor-neuron. Moreover, the survival of the injected AT- MSCs lasted for at least 14 days. These results indicate that neuronal survival after lesion, followed by mesenchymal stem cell (MSC) administration, might occur through cytokine release and immunomodulation, thus suggesting that AT-MSCs are promising cells for the therapy of neuronal lesions.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

OBJECTIVE: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. METHODS: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation. RESULTS: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals. INTERPRETATION: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.