Clickable Granular Hydrogel Scaffolds for Delivery of Neural Progenitor Cells to Sites of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious condition with limited treatment options. Neural progenitor cell (NPC) transplantation is a promising treatment option, and the identification of novel biomaterial scaffolds that support NPC engraftment and therapeutic activity is a top research priority. The objective of this study is to evaluate in situ assembled poly (ethylene glycol) (PEG)-based granular hydrogels for NPC delivery in a murine model of SCI. Microgel precursors are synthesized by using thiol-norbornene click chemistry to react four-armed PEG-amide-norbornene with enzymatically degradable and cell adhesive peptides. Unreacted norbornene groups are utilized for in situ assembly into scaffolds using a PEG-di-tetrazine linker. The granular hydrogel scaffolds exhibit good biocompatibility and do not adversely affect the inflammatory response after SCI. Moreover, when used to deliver NPCs, the granular hydrogel scaffolds supported NPC engraftment, do not adversely affect the immune response to the NPC grafts, and successfully support graft differentiation toward neuronal or astrocytic lineages as well as axonal extension into the host tissue. Collectively, these data establish PEG-based granular hydrogel scaffolds as a suitable biomaterial platform for NPC delivery and justify further testing, particularly in the context of more severe SCI.

Combinatorial strategies for cell transplantation in traumatic spinal cord injury.

Spinal cord injury (SCI) substantially reduces the quality of life of affected individuals. Recovery of function is therefore a primary concern of the patient population and a primary goal for therapeutic interventions. Currently, even with growing numbers of clinical trials, there are still no effective treatments that can improve neurological outcomes after SCI. A large body of work has demonstrated that transplantation of neural stem/progenitor cells (NSPCs) can promote regeneration of the injured spinal cord by providing new neurons that can integrate into injured host neural circuitry. Despite these promising findings, the degree of functional recovery observed after NSPC transplantation remains modest. It is evident that treatment of such a complex injury cannot be addressed with a single therapeutic approach. In this mini-review, we discuss combinatorial strategies that can be used along with NSPC transplantation to promote spinal cord regeneration. We begin by introducing bioengineering and neuromodulatory approaches, and highlight promising work using these strategies in integration with NSPCs transplantation. The future of NSPC transplantation will likely include a multi-factorial approach, combining stem cells with biomaterials and/or neuromodulation as a promising treatment for SCI.

Chemogenetic Attenuation of Acute Nociceptive Signaling Enhances Functional Outcomes Following Spinal Cord Injury.

Identifying novel therapeutic approaches to promote recovery of neurological functions following spinal cord injury (SCI) remains a great unmet need. Nociceptive signaling in the acute phase of SCI has been shown to inhibit recovery of locomotor function and promote the development of chronic neuropathic pain. We therefore hypothesized that inhibition of nociceptive signaling in the acute phase of SCI might improve long-term functional outcomes in the chronic phase of injury. To test this hypothesis, we took advantage of a selective strategy utilizing AAV6 to deliver inhibitory (hM4Di) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to nociceptors of the L4-L6 dorsal root ganglia to evaluate the effects of transient nociceptor silencing on long-term sensory and motor functional outcomes in a rat thoracic contusion SCI model. Following hM4Di-mediated nociceptor inhibition from 0-14 days post-SCI, we conducted behavioral assessments until 70 days post-SCI, then performed histological assessments of lesion severity and axon plasticity. Our results show highly selective expression of hM4Di within small diameter nociceptors including calcitonin gene-related peptide (CGRP)+ and IB4-binding neurons. Expression of hM4Di in less than 25% of nociceptors was sufficient to increase hindlimb thermal withdrawal latency in naïve rats. Compared with subjects who received AAV-yellow fluorescent protein (YFP; control), subjects who received AAV-hM4Di exhibited attenuated thermal hyperalgesia, greater coordination, and improved hindlimb locomotor function. However, treatment did not impact the development of cold allodynia or mechanical hyperalgesia. Histological assessments of spinal cord tissue suggested trends toward reduced lesion volume, increased neuronal sparing and increased CGRP+ axon sprouting in hM4Di-treated animals. Together, these findings suggest that nociceptor silencing early after SCI may promote beneficial plasticity in the acute phase of injury that can impact long-term functional outcomes, and support previous work highlighting primary nociceptors as possible therapeutic targets for pain management after SCI.

Differences in Anatomical Outcomes Between Early Chronic and Far Chronic Time-Points After Transplantation of Spinal Cord Neural Progenitor Cells in Mice.

Spinal cord injury (SCI) affects millions of people worldwide. Neural progenitor cell (NPC) transplantation is a promising treatment for regenerating lost spinal cord tissue and restoring neurological function after SCI. We conducted a literature search and found that less than a quarter of experimental rodent cell and tissue transplantation studies have investigated anatomical outcomes at longer than 4 months post-transplantation. This is a critical topic to investigate, given that stem and progenitor cell therapies would need to remain in place throughout the lifetime of an individual. We sought to determine how commonly assessed anatomical outcomes evolve between early and far chronic time-points post-NPC transplantation. At either 8 weeks or 26 weeks following transplantation of NPCs into sites of cervical SCI, we evaluated graft neuronal density, astroglial cell density, graft axon outgrowth, and regeneration of host axon populations into grafts in male and female mice. We found that graft neuronal density does not change over time, but the numbers of graft-associated astrocytes and glial fibrillary acidic protein intensity is significantly increased in the far chronic phase compared with the early chronic time-point. In addition, graft axon outgrowth was significantly decreased at 26 weeks post-transplantation compared with 8 weeks post-transplantation. In contrast, corticospinal axon regeneration into grafts was not diminished over time, but rather increased significantly from early to far chronic periods. Interestingly, we found that graft neuronal density is significantly influenced by sex of the host animal, suggesting that sex-dependent processes may shape graft composition over time. Collectively, these results demonstrate that NPC transplants are dynamic and that commonly assessed outcome measures associated with graft efficacy evolve over the weeks to months post-transplantation into the spinal cord.

Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Tempo and Mode of Genome Structure Evolution in Insects.

The division of the genome into discrete chromosomes is a fundamental characteristic of eukaryotic life. Insect taxonomists' early adoption of cytogenetics has led to an incredible amount of data describing genome structure across insects. In this article, we synthesize data from thousands of species and use biologically realistic models to infer the tempo and mode of chromosome evolution among insect orders. Our results show that orders vary dramatically in the overall rate of chromosome number evolution (a proxy of genome structural stability) and the pattern of evolution (e.g., the balance between fusions and fissions). These findings have important implications for our understanding of likely modes of speciation and offer insight into the most informative clades for future genome sequencing.

The Emerging Role of Biological Sex in Cell Therapy for Spinal Cord Injury.

Neural progenitor cell (NPC) transplantation is a promising potential therapy for replacing spinal cord neurons and glial cells following spinal cord injury (SCI). Despite the rapid advancement of NPC transplantation to SCI clinical trials, we still lack understanding of fundamental biology underlying how NPC grafts interact with the injured host nervous system. Our recent study demonstrated a potent effect of biological sex mismatch between donor and host on graft immune rejection. Here we discuss the implications of this study in the context of clinical trials for SCI, and important topics for future research in SCI cell transplantation.

Fighting for recovery on multiple fronts: The past, present, and future of clinical trials for spinal cord injury.

Through many decades of preclinical research, great progress has been achieved in understanding the complex nature of spinal cord injury (SCI). Preclinical research efforts have guided and shaped clinical trials, which are growing in number by the year. Currently, 1,149 clinical trials focused on improving outcomes after SCI are registered in the U.S. National Library of Medicine at ClinicalTrials.gov. We conducted a systematic analysis of these SCI clinical trials, using publicly accessible data downloaded from ClinicalTrials.gov. After extracting all available data for these trials, we categorized each trial according to the types of interventions being tested and the types of outcomes assessed. We then evaluated clinical trial characteristics, both globally and by year, in order to understand the areas of growth and change over time. With regard to clinical trial attributes, we found that most trials have low enrollment, only test single interventions, and have limited numbers of primary outcomes. Some gaps in reporting are apparent; for instance, over 75% of clinical trials with "Completed" status do not have results posted, and the Phase of some trials is incorrectly classified as "Not applicable" despite testing a drug or biological compound. When analyzing trials based on types of interventions assessed, we identified the largest representation in trials testing rehab/training/exercise, neuromodulation, and behavioral modifications. Most highly represented primary outcomes include motor function of the upper and lower extremities, safety, and pain. The most highly represented secondary outcomes include quality of life and pain. Over the past 15 years, we identified increased representation of neuromodulation and rehabilitation trials, and decreased representation of drug trials. Overall, the number of new clinical trials initiated each year continues to grow, signifying a hopeful future for the clinical treatment of SCI. Together, our work provides a comprehensive glimpse into the past, present, and future of SCI clinical trials, and suggests areas for improvement in clinical trial reporting.

Transcription Factor Hb9 Is Expressed in Glial Cell Lineages in the Developing Mouse Spinal Cord.

Hb9 (Mnx1) is a transcription factor described as a spinal cord motor neuron (MN)-specific marker and critical factor for the postmitotic specification of these cells. To date, expression of Hb9 in other cell types has not been reported. We performed a fate-mapping approach to examine distributions of Hb9-expressing cells and their progeny ("Hb9-lineage cells") within the embryonic and adult spinal cord of Hb9cre;Ai14 mice. We found that Hb9-lineage cells are distributed in a gradient of increasing abundance throughout the rostrocaudal spinal cord axis during embryonic and postnatal stages. Furthermore, although the majority of Hb9-lineage cells at cervical spinal cord levels are MNs, at more caudal levels, Hb9-lineage cells include small-diameter dorsal horn neurons, astrocytes, and oligodendrocytes. In the peripheral nervous system, we observed a similar phenomenon with more abundant Hb9-lineage Schwann cells in muscles of the lower body versus upper body muscles. We cultured spinal cord progenitors in vitro and found that gliogenesis was increased by treatment with the caudalizing factor FGF-8B, while glial tdTomato expression was increased by treatment with both FGF-8B and GDF-11. Together, these observations suggest that early and transient expression of Hb9 in spinal cord neural progenitors may be induced by caudalizing factors such as FGF and GDF signaling. Furthermore, our work raises the possibility that early Hb9 expression may influence the development of spinal cord macroglia and Schwann cells, especially at caudal regions. Together, these findings highlight the importance of using caution when designing experiments using Hb9cre mice to perform spinal cord MN-specific manipulations.

Effects of biological sex mismatch on neural progenitor cell transplantation for spinal cord injury in mice.

Despite advancement of neural progenitor cell transplantation to spinal cord injury clinical trials, there remains a lack of understanding of how biological sex of transplanted cells influences outcomes after transplantation. To address this, we transplanted GFP-expressing sex-matched, sex-mismatched, or mixed donor cells into sites of spinal cord injury in adult male and female mice. Biological sex of the donor cells does not influence graft neuron density, glial differentiation, formation of the reactive glial cell border, or graft axon outgrowth. However, male grafts in female hosts feature extensive hypervascularization accompanied by increased vascular diameter and perivascular cell density. We show greater T-cell infiltration within male-to-female grafts than other graft types. Together, these findings indicate a biological sex-specific immune response of female mice to male donor cells. Our work suggests that biological sex should be considered in the design of future clinical trials for cell transplantation in human injury.

Dorsal horn neuronal sparing predicts the development of at-level mechanical allodynia following cervical spinal cord injury in mice.

Spinal cord injury (SCI) frequently results in immediate and sustained neurological dysfunction, including intractable neuropathic pain in approximately 60-80% of individuals. SCI induces immediate mechanical damage to spinal cord tissue followed by a period of secondary injury in which tissue damage is further propagated, contributing to the development of anatomically unique lesions. Variability in lesion size and location influences the degree of motor and sensory dysfunction incurred by an individual. We predicted that variability in lesion parameters may also explain why some, but not all, experimental animals develop mechanical sensitivity after SCI. To characterize the relationship of lesion anatomy to mechanical allodynia, we utilized a mouse cervical hemicontusion model of SCI that has been shown to lead to the development and persistence of mechanical allodynia in the ipsilateral forelimb after injury. At four weeks post-SCI, the numbers and locations of surviving neurons were quantified along with total lesion volume and nociceptive fiber sprouting. We found that the subset of animals exhibiting mechanical allodynia had significantly increased neuronal sparing in the ipsilateral dorsal horn around the lesion epicenter compared to animals that did not exhibit mechanical allodynia. Additionally, we failed to observe significant differences between groups in nociceptive fiber density in the dorsal horn around the lesion epicenter. Notably, we found that impactor probe displacement upon administration of the SCI surgery was significantly lower in sensitive animals compared with not-sensitive animals. Together, our data indicate that lesion severity negatively correlates with the manifestation of at-level mechanical hypersensitivity and suggests that sparing of dorsal horn neurons may be required for the development of neuropathic pain.

An Analysis of Variability in "CatWalk" Locomotor Measurements to Aid Experimental Design and Interpretation.

Preclinical studies in models of neurologic injury and disease rely on behavioral outcomes to measure intervention efficacy. For spinal cord injury, the CatWalk system provides unbiased quantitative assessment of subtle aspects of locomotor function in rodents and so can powerfully detect significant differences between experimental and control groups. Although clearly of key importance, summary group-level data can obscure the variability within and between individual subjects and therefore make it difficult to understand the magnitude of effect in individual animals and the proportion of a group that may show benefit. Here, we calculate reference change intervals (RCIs) that define boundaries of normal variability for measures of rat locomotion on the CatWalk. Our results indicate that many commonly-used outcome measures are highly variable, such that differences of up to 70% from baseline value must be considered normal variation. Many CatWalk outcome variables are also highly correlated and dependent on run speed. Application of calculated RCIs to open access data (https://scicrunch.org/odc-sci) on hindlimb stride length in spinal cord-injured rats illustrates the complementarity between group-level (16 mm change; p = 0.0009) and individual-level (5/32 animals show change outside RCI boundaries) analysis between week 3 and week 6 after injury. We also conclude that interdependence among CatWalk variables implies that test "batteries" require careful composition to ensure that different aspects of defective gait are analyzed. Calculation of RCIs aids in experimental design by quantifying variability and enriches overall data analysis by providing details of change at an individual level that complement group-level analysis.

Transplanting neural progenitor cells to restore connectivity after spinal cord injury.

Spinal cord injury remains a scientific and therapeutic challenge with great cost to individuals and society. The goal of research in this field is to find a means of restoring lost function. Recently we have seen considerable progress in understanding the injury process and the capacity of CNS neurons to regenerate, as well as innovations in stem cell biology. This presents an opportunity to develop effective transplantation strategies to provide new neural cells to promote the formation of new neuronal networks and functional connectivity. Past and ongoing clinical studies have demonstrated the safety of cell therapy, and preclinical research has used models of spinal cord injury to better elucidate the underlying mechanisms through which donor cells interact with the host and thus increase long-term efficacy. While a variety of cell therapies have been explored, we focus here on the use of neural progenitor cells obtained or derived from different sources to promote connectivity in sensory, motor and autonomic systems.

Intraspinal and Intracortical Delivery of AAV Vectors for Intersectional Circuit Tracing in Non-transgenic Species.

The mapping of long-range axonal projections is a rapidly growing endeavor in the field of neuroscience. Recent advances in the development of adeno-associated viral vectors that can achieve efficient retrograde transport now enable the characterization and manipulation of specific neuronal subpopulations using Cre-dependent, intersectional approaches. Importantly, these approaches can be applied to non-transgenic animals, making it possible to carry out detailed anatomical studies across a variety of species including nonhuman primates. In this chapter, we demonstrate the utility of such intersectional strategies by describing methods for targeting viral constructs to distinct subsets of corticospinal motor neurons based on their projections to specific spinal cord segments.

Biomimetic 3D-printed scaffolds for spinal cord injury repair.

Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation1. 3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (µCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. µCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents1,2. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.

Adult rat myelin enhances axonal outgrowth from neural stem cells.

Axon regeneration after spinal cord injury (SCI) is attenuated by growth inhibitory molecules associated with myelin. We report that rat myelin stimulated the growth of axons emerging from rat neural progenitor cells (NPCs) transplanted into sites of SCI in adult rat recipients. When plated on a myelin substrate, neurite outgrowth from rat NPCs and from human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) was enhanced threefold. In vivo, rat NPCs and human iPSC-derived NSCs extended greater numbers of axons through adult central nervous system white matter than through gray matter and preferentially associated with rat host myelin. Mechanistic investigations excluded Nogo receptor signaling as a mediator of stem cell-derived axon growth in response to myelin. Transcriptomic screens of rodent NPCs identified the cell adhesion molecule neuronal growth regulator 1 (Negr1) as one mediator of permissive axon-myelin interactions. The stimulatory effect of myelin-associated proteins on rodent NPCs was developmentally regulated and involved direct activation of the extracellular signal-regulated kinase (ERK). The stimulatory effects of myelin on NPC/NSC axon outgrowth should be investigated further and could potentially be exploited for neural repair after SCI.

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.

Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA.