Neural Grafts Research Articles

The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration.

Over the past decade, we have witnessed a flourishing of novel strategies to enhance neuroplasticity and promote axon regeneration following spinal cord injury, and results from preclinical studies suggest that some of these strategies have the potential for clinical translation. Spinal cord injury leads to the disruption of neural circuitry and connectivity, resulting in permanent neurological disability. Recovery of function relies on augmenting neuroplasticity topotentiate sprouting and regeneration of spared and injured axons, to increase the strength ofresidual connections and to promote the formation of new connections and circuits. Neuroplasticity can be fostered by exploiting four main biological properties: neuronal intrinsic signalling, the neuronal extrinsic environment, the capacity to reconnect the severed spinal cord via neural stem cell grafts, and modulation of neuronal activity. In this Review, we discuss experimental evidence from rodents, nonhuman primates and patients regarding interventions that target each of these four properties. We then highlight the strengths and challenges of individual and combinatorial approaches with respect to clinical translation. We conclude byconsidering future developments and providing views on how to bridge the gap between preclinical studies and clinical translation.

Monitoring maturation of neural stem cell grafts within a host microenvironment.

Neural stem cells (NSC) act as a versatile tool for neuronal cell replacement strategies to treat neurodegenerative disorders in which functional neurorestorative mechanisms are limited. While the beneficial effects of such cell-based therapy have already been documented in terms of neurodegeneration of various origins, a neurophysiological basis for improvement in the recovery of neurological function is still not completely understood. This overview briefly describes the cumulative evidence from electrophysiological studies of NSC-derived neurons, aimed at establishing the maturation of differentiated neurons within a host microenvironment, and their integration into the host circuits, with a particular focus on the neurogenesis of NSC grafts within the post-ischemic milieu. Overwhelming evidence demonstrates that the host microenvironment largely regulates the lineage of NSC grafts. This regulatory role, as yet underestimated, raises possibilities for the favoured maturation of a subset of neural phenotypes in order to gain timely remodelling of the impaired brain tissue and amplify the therapeutic effects of NSC-based therapy for recovery of neurological function.

MHC matching fails to prevent long-term rejection of iPSC-derived neurons in non-human primates

Cell therapy products (CTP) derived from pluripotent stem cells (iPSCs) may constitute a renewable, specifically differentiated source of cells to potentially cure patients with neurodegenerative disorders. However, the immunogenicity of CTP remains a major issue for therapeutic approaches based on transplantation of non-autologous stem cell-derived neural grafts. Despite its considerable side-effects, long-term immunosuppression, appears indispensable to mitigate neuro-inflammation and prevent rejection of allogeneic CTP. Matching iPSC donors’ and patients’ HLA haplotypes has been proposed as a way to access CTP with enhanced immunological compatibility, ultimately reducing the need for immunosuppression. In the present work, we challenge this paradigm by grafting autologous, MHC-matched and mis-matched neuronal grafts in a primate model of Huntington’s disease. Unlike previous reports in unlesioned hosts, we show that in the absence of immunosuppression MHC matching alone is insufficient to grant long-term survival of neuronal grafts in the lesioned brain.

Tissue-Engineered Neural Network Graft Relays Excitatory Signal in the Completely Transected Canine Spinal Cord.

Tissue engineering produces constructs with defined functions for the targeted treatment of damaged tissue. A complete spinal cord injury (SCI) model is generated in canines to test whether in vitro constructed neural network (NN) tissues can relay the excitatory signal across the lesion gap to the caudal spinal cord. Established protocols are used to construct neural stem cell (NSC)‐derived NN tissue characterized by a predominantly neuronal population with robust trans‐synaptic activities and myelination. The NN tissue is implanted into the gap immediately following complete transection SCI of canines at the T10 spinal cord segment. The data show significant motor recovery of paralyzed pelvic limbs, as evaluated by Olby scoring and cortical motor evoked potential (CMEP) detection. The NN tissue survives in the lesion area with neuronal phenotype maintenance, improves descending and ascending nerve fiber regeneration, and synaptic integration with host neural circuits that allow it to serve as a neuronal relay to transmit excitatory electrical signal across the injured area to the caudal spinal cord. These results suggest that tissue‐engineered NN grafts can relay the excitatory signal in the completely transected canine spinal cord, providing a promising strategy for SCI treatment in large animals, including humans.

Roles of Reelin/Disabled1 pathway on functional recovery of hemiplegic mice after neural cell transplantation; Reelin promotes migration toward motor cortex and maturation to motoneurons of neural grafts

Reelin is a large glycoprotein which regulates central nervous system (CNS) development. Dysfunctions of Reelin were reported on certain neuropsychiatric diseases. We examined involvement of Reelin pathway in functional recovery of hemiplegic mice after neural transplantation.Reelin was expressed 1 day after cryogenic injury of right motor cortex. We transplanted neural stem/progenitor cells (NSPCs) from wild-type mice into ipsilateral striatum of hemiplegic mice. The grafts migrated from the striatum and reached the injured cortex 14 days after transplantation. The transplantation significantly improved their motor functions (P < .05).The NSPCs migrating toward the cortex expressed Reelin receptors, Apoer and Vldlr, and phosphorylated Disabled1 (Dab1), a downstream signaling molecule of Reelin. The grafts expressed Ncadherin and active form of Integrin β1, both of which were known to become active with Reelin stimulation. At day 28, the grafts expressed Ctip2, Crim1, Foxp2, and Fezf2, all of which were forebrain motoneuron associated markers, and Nfm and Synapsin1 on the damaged cortex.We then transplanted NSPCs of yotari mice (yot/yot genotype) having nonfunctional Dab1 by a mutation of its gene. Majority of the grafts from yotari mice (>80%) did not migrate and thus remained at the striatum. The grafts did not express the forebrain motoneuron associated markers nor the cell adhesion molecules including Ncadherin and active Integrin β1.Reelin pathway was involved in graft migration by regulating certain adhesion molecules and in their differentiation to functional motoneurons accompanying synapse formation. We suggested involvement of Reelin pathway for neural regeneration and functional recovery of hemiplegic mice in adulthood after neural transplantation.

SUMOylation promotes survival and integration of neural stem cell grafts in ischemic stroke.

BackgroundNeural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments.MethodsHerein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion.FindingsNSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo.InterpretationOur work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke.FundIntramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.

Regenerating Corticospinal Axons Innervate Phenotypically Appropriate Neurons within Neural Stem Cell Grafts

SUMMARYNeural progenitor cell grafts form new relays across sites of spinal cord injury (SCI). Using a panel of neuronal markers, we demonstrate that spinal neural progenitor grafts to sites of rodent SCI adopt diverse spinal motor and sensory interneuronal fates, representing most neuronal subtypes of the intact spinal cord, and spontaneously segregate into domains of distinct cell clusters. Host corticospinal motor axons regenerating into neural progenitor grafts innervate appropriate pre-motor interneurons, based on trans-synaptic tracing with herpes simplex virus. A human spinal neural progenitor cell graft to a non-human primate also received topographically appropriate corticospinal axon regeneration. Thus, grafted spinal neural progenitor cells give rise to a variety of neuronal progeny that are typical of the normal spinal cord; remarkably, regenerating injured adult corticospinal motor axons spontaneously locate appropriate motor domains in the heterogeneous, developing graft environment, without a need for additional exogenous guidance.

Astrocytes migrate from human neural stem cell grafts and functionally integrate into the injured rat spinal cord

Neural stem cells (NSCs) can differentiate into both neurons and glia after transplantation into spinal cord injury (SCI) sites. The neuronal component of stem cell grafts has the potential to form functional synaptic relays across the lesion site. The glial component may reform a blood-spinal cord barrier, support neuronal function, and contribute to remyelination. We performed a long-term, 1.5-year time course study focused on astrocyte migration, differentiation, integration, and safety following human NSC transplantation into C5 hemisection sites in immunodeficient rats. NSCs that adopted a neuronal fate did not migrate from the lesion site. In contrast, transplanted cells that adopted astrocyte fates exhibited long distance migration from the lesion site through host white matter in rostrocaudal directions. These cells migrated slowly at a mean rate of 2–3 mm/month, divided as they migrated, and gradually differentiated into astrocytes. After 1.5 years, human astrocytes migrated nine spinal cord segments, caudally to the mid-thoracic level, and rostrally into the brainstem. The migrated human astrocytes joined the endogenous population of astrocytes in the host spinal cord, extended perivascular endfeet towards host pericytes and endothelium, formed interspecies and intraspecies perivascular astrocytic networks connected by gap junctions, and expressed glutamate transporter proteins in perisynaptic processes, suggesting structural and functional integration. No adverse consequences of this extended glial migration were detected. Adjacent to the lesion site, chronic host astrocytic upregulation was significantly attenuated by NSC grafts. Thus, human astrocytes can migrate long distances from sites of SCI and safely integrate into the host central nervous system. Significance statementNeural stem cell (NSC) grafts are a candidate therapy for spinal cord injury (SCI). Here we report an 18-month study of astrocyte survival and migration from sites of SCI in immunodeficient rats. NSC grafts significantly attenuate host astrocyte reactivity at the lesion/host interface. Intra-graft astrocytes integrate into the host blood-spinal cord barrier (BSCB) and widely express glutamate transporter proteins characteristic of neurotransmitter regulation. Notably, astrocytic components of NSC grafts exhibit gradual yet extensive migration after implantation into the mid-cervical injury site; neurons do not migrate at all. This extensive astrocyte migration is not detectably associated with adverse outcomes anatomically or behaviorally.

Human parthenogenetic neural stem cell grafts promote multiple regenerative processes in a traumatic brain injury model.

International Stem Cell Corporation human parthenogenetic neural stem cells (ISC-hpNSC) have potential therapeutic value for patients suffering from traumatic brain injury (TBI). Here, we demonstrate the behavioral and histological effects of transplanting ISC-hpNSC intracerebrally in an animal model of TBI.Methods: Sprague-Dawley rats underwent a moderate controlled cortical impact TBI surgery. Transplantation occurred at 72 h post-TBI with functional readouts of behavioral and histological deficits conducted during the subsequent 3-month period after TBI. We characterized locomotor, neurological, and cognitive performance at baseline (before TBI), then on days 0, 1, 7, 14, 30, 60, and 90 (locomotor and neurological), and on days 28-30, 58-60, and 88-90 (cognitive) after TBI. Following completion of behavioral testing at 3 months post-TBI, animals were euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of brain damage. Neuronal survival was revealed by Nissl staining, and stem cell engraftment and host tissue repair mechanisms such as the anti-inflammatory response in peri-TBI lesion areas were examined by immunohistochemical analyses.Results: We observed that TBI groups given high and moderate doses of ISC-hpNSC had an improved swing bias on an elevated body swing test for motor function, increased scores on forelimb akinesia and paw grasp neurological tests, and committed significantly fewer errors on a radial arm water maze test for cognition. Furthermore, histological analyses indicated that high and moderate doses of stem cells increased the expression of phenotypic markers related to the neural lineage and myelination and decreased reactive gliosis and inflammation in the brain, increased neuronal survival in the peri-impact area of the cortex, and decreased inflammation in the spleen at 90 days post-TBI.Conclusion: These results provide evidence that high and moderate doses of ISC-hpNSC ameliorate TBI-associated histological alterations and motor, neurological, and cognitive deficits.

Insulin-like Growth Factor-1 Receptor Dictates Beneficial Effects of Treadmill Training by Regulating Survival and Migration of Neural Stem Cell Grafts in the Injured Spinal Cord.

Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/−) for Igf1r, the gene encoding IGF-1 receptor, into the mouse spinal cord after injury, with or without combining TMT. We analyzed the influence of genotype and TMT on locomotor recovery and survival and migration of NSC grafts. In vitro experiments were performed to examine the potential roles of IGF-1 signaling in the migratory ability of NSCs. Mice receiving +/− NSC grafts showed impaired locomotor recovery compared with those receiving wild-type (+/+) NSCs. Locomotor improvement by TMT was more pronounced with +/+ grafts. Deficiency of one allele of Igf1r significantly reduced survival and migration of the transplanted NSCs. Although TMT did not significantly influence NSC survival, it substantially enhanced the extent of migration for only +/+ NSCs. Cultured neurospheres exhibited dynamic motility with cytoplasmic protrusions, which was regulated by IGF-1 signaling. IGF-1 signaling in transplanted NSCs may be essential in regulating their survival and migration. Furthermore, TMT may promote NSC graft-mediated locomotor recovery via activation of IGF-1 signaling in transplanted NSCs. Dynamic NSC motility via IGF-1 signaling may be the cellular basis for the TMT-induced enhancement of migration.

Activation of Intrinsic Growth State Enhances Host Axonal Regeneration into Neural Progenitor Cell Grafts.

SummaryAxonal regeneration after spinal cord injury (SCI) can be enhanced by activation of the intrinsic neuronal growth state and, separately, by placement of growth-enabling neural progenitor cell (NPC) grafts into lesion sites. Indeed, NPC grafts support regeneration of all host axonal projections innervating the normal spinal cord. However, some host axons regenerate only short distances into grafts. We examined whether activation of the growth state of the host injured neuron would elicit greater regeneration into NPC grafts. Rats received NPC grafts into SCI lesions in combination with peripheral “conditioning” lesions. Six weeks later, conditioned host sensory axons exhibited a significant, 9.6-fold increase in regeneration into the lesion/graft site compared with unconditioned axons. Regeneration was further enhanced 1.6-fold by enriching NPC grafts with phenotypically appropriate sensory neuronal targets. Thus, activation of the intrinsic host neuronal growth state and manipulation of the graft environment enhance axonal regeneration after SCI.

Restoration of damaged cortical pathways by neural grafting

The motor cortex plays a central role in the control, planning, and execution of voluntary motor commands in mammals. The loss of cortical neurons is a common feature of many neuropathological conditions such as traumatic and ischemic lesions or several neurodegenerative diseases. Cell transplantation presents a promising therapeutic strategy to overcome the limited abilities of axonal regrowth and spontaneous regeneration of the adult central nervous system. In this review, we will present a historical review of brain transplantation and the current state of research in the field of cortical transplantation.

Genetically Corrected iPSC-Derived Neural Stem Cell Grafts Deliver Enzyme Replacement to Affect CNS Disease in Sanfilippo B Mice

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB [MPS IIIB]) is a lysosomal storage disorder primarily affecting the brain that is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. Here we report that, ex vivo, lentiviral correction of Naglu−/− neural stem cells derived from Naglu−/− mice (iNSCs) corrected their lysosomal pathology and allowed them to secrete a functional NAGLU enzyme that could be taken up by deficient cells. Following long-term transplantation of these corrected iNSCs into Naglu−/− mice, we detected NAGLU activity in the majority of engrafted animals. Successfully transplanted Naglu−/− mice showed a significant decrease in storage material, a reduction in astrocyte activation, and complete prevention of microglial activation within the area of engrafted cells and neighboring regions, with beneficial effects extending partway along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu−/− mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver enzymes and treat MPS IIIB.

Brain Region-Dependent Rejection of Neural Precursor Cell Transplants.

The concept of CNS as an immune-privileged site has been challenged by the occurrence of immune surveillance and allogeneic graft rejection in the brain. Here we examined whether the immune response to allogeneic neural grafts is determined by the site of implantation in the CNS. Dramatic regional differences were observed between immune responses to allogeneic neural precursor/stem cell (NPC) grafts in the striatum vs. the hippocampus. Striatal grafts were heavily infiltrated with IBA-1+ microglia/macrophages and CD3+ T cells and completely rejected. In contrast, hippocampal grafts exhibited milder IBA-1+ cell infiltration, were not penetrated efficiently by CD3+ cells, and survived efficiently for at least 2 months. To evaluate whether the hippocampal protective effect is universal, astrocytes were then transplanted. Allogeneic astrocyte grafts elicited a vigorous rejection process from the hippocampus. CD200, a major immune-inhibitory signal, plays an important role in protecting grafts from rejection. Indeed, CD200 knock out NPC grafts were rejected more efficiently than wild type NPCs from the striatum. However, lack of CD200 expression did not elicit NPC graft rejection from the hippocampus. In conclusion, the hippocampus has partial immune-privilege properties that are restricted to NPCs and are CD200-independent. The unique hippocampal milieu may be protective for allogeneic NPC grafts, through host-graft interactions enabling sustained immune-regulatory properties of transplanted NPCs. These findings have implications for providing adequate immunosuppression in clinical translation of cell therapy.

Neural Stem Cell Grafts Promote Astroglia-Driven Neurorestoration in the Aged Parkinsonian Brain via Wnt/β-Catenin Signaling

During aging-one the most potent risk factors for Parkinson's disease (PD)-both astrocytes and microglia undergo functional changes that ultimately hamper homoeostasis, defense, and repair of substantia nigra pars compacta (SNpc) midbrain dopaminergic (mDA) neurons. We tested the possibility of rejuvenating the host microenvironment and boosting SNpc DA neuronal plasticity via the unilateral transplantation of syngeneic neural stem/progenitor cells (NSCs) in the SNpc of aged mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental PD. Transplanted NSCs within the aged SNpc engrafted and migrated in large proportions to the tegmental aqueduct mDA niche, with 30% acquiring an astroglial phenotype. Both graft-derived exogenous (ex-Astro) and endogenous astrocytes (en-Astro) expressed Wnt1. Both ex-Astro and en-Astro were key triggers of Wnt/β-catenin signaling in SNpc-mDA neurons and microglia, which was associated with mDA neurorescue and immunomodulation. At the aqueduct-ventral tegmental area level, NSC grafts recapitulated a genetic Wnt1-dependent mDA developmental program, inciting the acquisition of a mature Nurr1+ TH+ neuronal phenotype. Wnt/β-catenin signaling antagonism abolished mDA neurorestoration and immune modulatory effects of NSC grafts. Our work implicates an unprecedented therapeutic potential for somatic NSC grafts in the restoration of mDA neuronal function in the aged Parkinsonian brain. Stem Cells 2018;36:1179-1197.

Spinal cord: Making new connections.

This study shows that human neural stem cell grafts re-establish a neuronal relay across spinal cord injury in rhesus monkeys.

Sensors of Succinate: Neural Stem Cell Grafts Fight Neuroinflammation.

In this issue of Cell Stem Cell, Peruzzotti-Jametti etal. (2018) demonstrate how neural stem cells, transplanted in a mouse model of multiple sclerosis, respond to extracellular succinate and modulate neuroinflammation by releasing anti-inflammatory prostaglandin E2 and scavenging succinate. This mechanism reduces CNS damage and ameliorates motor impairment.

Restorative effects of human neural stem cell grafts on the primate spinal cord.

We grafted human spinal cord-derived neural progenitor cells (NPCs) into sites of cervical spinal cord injury in rhesus monkeys (Macaca mulatta). Under three-drug immunosuppression, grafts survived at least 9 months postinjury and expressed both neuronal and glial markers. Monkey axons regenerated into grafts and formed synapses. Hundreds of thousands of human axons extended out from grafts through monkey white matter and synapsed in distal gray matter. Grafts gradually matured over 9 months and improved forelimb function beginning several months after grafting. These findings in a 'preclinical trial' support translation of NPC graft therapy to humans with the objective of reconstituting both a neuronal and glial milieu in the site of spinal cord injury.

Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts

Neural progenitor cell (NPC) transplantation has high therapeutic potential in neurological disorders. Functional restoration may depend on the formation of reciprocal connections between host and graft. While it has been reported that axons extending out of neural grafts in the brain form contacts onto phenotypically appropriate host target regions, it is not known whether adult, injured host axons regenerating into NPC grafts also form appropriate connections. We report that spinal cord NPCs grafted into the injured adult rat spinal cord self-assemble organotypic, dorsal horn-like domains. These clusters are extensively innervated by regenerating adult host sensory axons and are avoided by corticospinal axons. Moreover, host axon regeneration into grafts increases significantly after enrichment with appropriate neuronal targets. Together, these findings demonstrate that injured adult axons retain the ability to recognize appropriate targets and avoid inappropriate targets within neural progenitor grafts, suggesting that restoration of complex circuitry after SCI may be achievable.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

SummaryDevelopment of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.