Divergent transcriptional regulation of astrocyte reactivity across disorders.

Astrocytes respond to injury and disease in the central nervous system with reactive changes that influence the outcome of the disorder1-4. These changes include differentially expressed genes (DEGs) whose contextual diversity and regulation are poorly understood. Here we combined biological and informatic analyses, including RNA sequencing, protein detection, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and conditional gene deletion, to predict transcriptional regulators that differentially control more than 12,000 DEGs that are potentially associated with astrocyte reactivity across diverse central nervous system disorders in mice and humans. DEGs associated with astrocyte reactivity exhibited pronounced heterogeneity across disorders. Transcriptional regulators also exhibited disorder-specific differences, but a core group of 61 transcriptional regulators was identified as common across multiple disorders in both species. We show experimentally that DEG diversity is determined by combinatorial, context-specific interactions between transcriptional regulators. Notably, the same reactivity transcriptional regulators can regulate markedly different DEG cohorts in different disorders; changes in the access of transcriptional regulators to DNA-binding motifs differ markedly across disorders; and DEG changes can crucially require multiple reactivity transcriptional regulators. We show that, by modulating reactivity, transcriptional regulators can substantially alter disorder outcome, implicating them as therapeutic targets. We provide searchable resources of disorder-related reactive astrocyte DEGs and their predicted transcriptional regulators. Our findings show that transcriptional changes associated with astrocyte reactivity are highly heterogeneous and are customized from vast numbers of potential DEGs through context-specific combinatorial transcriptional-regulator interactions.

Required growth facilitators propel axon regeneration across complete spinal cord injury.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

Astrocyte scar formation aids central nervous system axon regeneration.

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

A new model for in vitro testing of vitreous substitute candidates.

PURPOSE: To describe a new model for in vitro assessment of novel vitreous substitute candidates. METHODS: The biological impact of three vitreous substitute candidates was explored in a retinal explant culture model; a polyalkylimide hydrogel (Bio-Alcamid®), a two component hydrogel of 20 wt.% poly (ethylene glycol) in phosphate buffered saline (PEG) and a cross-linked sodium hyaluronic acid hydrogel (Healaflow®). The gels where applied to explanted adult rat retinas and then kept in culture for 2, 5 and 10 days. Gel-exposed explants were compared with explants incubated under standard tissue culture conditions. Cryosections of the specimens were stained with hematoxylin and eosin, immunohistochemical markers (GFAP, Vimentin, Neurofilament 160, PKC, Rhodopsin) and TUNEL. RESULTS: Explants kept under standard conditions as well as PEG-exposed explants displayed disruption of retinal layers with moderate pyknosis of all neurons. They also displayed moderate labeling of apoptotic cells. Bio-Alcamid®-exposed explants displayed severe thinning and disruption of retinal layers with massive cell death. Healaflow®-treated explants displayed normal retinal lamination with significantly better preservation of retinal neurons compared with control specimens, and almost no signs of apoptosis. Retinas exposed to Healaflow® and retinas kept under standard conditions showed variable labeling of GFAP with generally low expression and some areas of upregulation. PEG-exposed retinas showed increased GFAP labeling and Bio-Alcamid®-exposed retinas showed sparse labeling of GFAP. CONCLUSIONS: Research into novel vitreous substitutes has important implications for both medical and surgical vitreoretinal disease. The in vitro model presented here provides a method of biocompatibility testing prior to more costly and cumbersome in vivo experiments. The explant culture system imposes reactions within the retina including disruption of layers, cell death and gliosis, and the progression of these reactions can be used for comparison of vitreous substitute candidates. Bio-Alcamid® had strong adverse effects on the retina which is consistent with results of prior in vivo trials. PEG gel elicits reactions similar to the control retinas whereas Healaflow® shows protection from culture-induced trauma indicating favorable biocompatibility.

Trehalose-based coacervates for local bioactive protein delivery to the central nervous system.

Therapeutic outcomes of local biomolecule delivery to the central nervous system (CNS) using bulk biomaterials are limited by inadequate drug loading, neuropil disruption, and severe foreign body responses. Effective CNS delivery requires addressing these issues and developing well-tolerated, highly-loaded carriers that are dispersible within local neural parenchyma. Here, we synthesized biodegradable trehalose-based polyelectrolyte oligomers using facile A2:B3:AR thiol-ene Michael addition reactions that form complex coacervates upon mixing of oppositely charged oligomers. Coacervates permit high concentration loading and controlled release of bioactive growth factors, enzymes, and antibodies, with modular formulation parameters that confer tunable release kinetics. Coacervates are cytocompatible with cultured neural cells in vitro and can be formulated to either direct intracellular protein delivery or sequester media containing proteins and remain extracellular. Coacervates serve as effective vehicles for precisely delivering biomolecules, including bioactive neurotrophins, to the mouse striatum following intraparenchymal injection. These results support the use of trehalose-based coacervates as part of therapeutic protein delivery strategies for CNS disorders.

Ischemic and hemorrhagic stroke lesion environments differentially alter the glia repair potential of neural progenitor cell and immature astrocyte grafts.

Using cell grafting to direct glia-based repair mechanisms in adult CNS injuries represents a potential therapeutic strategy for supporting functional neural parenchymal repair. However, glia repair directed by neural progenitor cell (NPC) grafts is dramatically altered by increasing lesion size, severity, and mode of injury. To address this, we studied the interplay between astrocyte differentiation and cell proliferation of NPC in vitro to generate proliferating immature astrocytes (ImA) using hysteretic conditioning. ImA maintain proliferation rates at comparable levels to NPC but showed robust immature astrocyte marker expression including Gfap and Vimentin. ImA demonstrated enhanced resistance to myofibroblast-like phenotypic transformations upon exposure to serum enriched environments in vitro compared to NPC and were more effective at scratch wound closure in vitro compared to quiescent astrocytes. Glia repair directed by ImA at acute ischemic striatal stroke lesions was equivalent to NPC but better than quiescent astrocyte grafts. While ischemic injury environments supported enhanced survival of grafts compared to healthy striatum, hemorrhagic lesions were hostile towards both NPC and ImA grafts leading to poor survival and ineffective modulation of natural wound repair processes. Our findings demonstrate that lesion environments, rather than transcriptional pre-graft states, determine the survival, cell-fate, and glia repair competency of cell grafts applied to acute CNS injuries.

Derivation and transcriptional reprogramming of border-forming wound repair astrocytes after spinal cord injury or stroke in mice.

Central nervous system (CNS) lesions become surrounded by neuroprotective borders of newly proliferated reactive astrocytes; however, fundamental features of these cells are poorly understood. Here we show that following spinal cord injury or stroke, 90% and 10% of border-forming astrocytes derive, respectively, from proliferating local astrocytes and oligodendrocyte progenitor cells in adult mice of both sexes. Temporal transcriptome analysis, single-nucleus RNA sequencing and immunohistochemistry show that after focal CNS injury, local mature astrocytes dedifferentiate, proliferate and become transcriptionally reprogrammed to permanently altered new states, with persisting downregulation of molecules associated with astrocyte-neuron interactions and upregulation of molecules associated with wound healing, microbial defense and interactions with stromal and immune cells. These wound repair astrocytes share morphologic and transcriptional features with perimeningeal limitans astrocytes and are the predominant source of neuroprotective borders that re-establish CNS integrity around lesions by separating neural parenchyma from stromal and immune cells as occurs throughout the healthy CNS.

Trehalose-Guanosine Glycopolymer Hydrogels Direct Adaptive Glia Responses in CNS Injury.

Neural tissue damaged after central nervous system (CNS) injury does not naturally regenerate but is instead replaced by non-neural fibrotic scar tissue that serves no neurological function. Scar-free repair to create a more permissive environment for regeneration requires altering the natural injury responses of glial cells. In this work, glycopolymer-based supramolecular hydrogels are synthesized to direct adaptive glia repair after CNS injury. Combining poly(trehalose-co-guanosine) (pTreGuo) glycopolymers with free guanosine (fGuo) generates shear-thinning hydrogels through stabilized formation of long-range G-quadruplex secondary structures. Hydrogels with smooth or granular microstructures and mechanical properties spanning three orders of magnitude are produced through facile control of pTreGuo hydrogel composition. Injection of pTreGuo hydrogels into healthy mouse brains elicits minimal stromal cell infiltration and peripherally derived inflammation that is comparable to a bioinert methyl cellulose benchmarking material. pTreGuo hydrogels alter astrocyte borders and recruit microglia to infiltrate and resorb the hydrogel bulk over 7 d. Injections of pTreGuo hydrogels into ischemic stroke alter the natural responses of glial cells after injury to reduce the size of lesions and increase axon regrowth into lesion core environments. These results support the use of pTreGuo hydrogels as part of neural regeneration strategies to activate endogenous glia repair mechanisms.

Proliferation history and transcription factor levels drive direct conversion.

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies.

Injectable diblock copolypeptide hydrogel provides platform to deliver effective concentrations of paclitaxel to an intracranial xenograft model of glioblastoma.

INTRODUCTION: Surgical resection and systemic chemotherapy with temozolomide remain the mainstay for treatment of glioblastoma. However, many patients are not candidates for surgical resection given inaccessible tumor location or poor health status. Furthermore, despite being first line treatment, temozolomide has only limited efficacy. METHODS: The development of injectable hydrogel-based carrier systems allows for the delivery of a wide range of chemotherapeutics that can achieve high local concentrations, thus potentially avoiding systemic side effects and wide-spread neurotoxicity. To test this modality in a realistic environment, we developed a diblock copolypeptide hydrogel (DCH) capable of carrying and releasing paclitaxel, a compound that we found to be highly potent against primary gliomasphere cells. RESULTS: The DCH produced minimal tissue reactivity and was well tolerated in the immune-competent mouse brain. Paclitaxel-loaded hydrogel induced less tissue damage, cellular inflammation and reactive astrocytes than cremaphor-taxol (typical taxol-carrier) or hydrogel alone. In a deep subcortical xenograft model of glioblastoma in immunodeficient mice, injection of paclitaxel-loaded hydrogel led to local tumor control and improved survival. However, the tumor cells were highly migratory and were able to eventually escape the area of treatment. CONCLUSIONS: These findings suggest this technology may be ultimately applicable to patients with deep-seated inoperable tumors, but as currently formulated, complete tumor eradication would be highly unlikely. Future studies should focus on targeting the migratory potential of surviving cells.

Cell biology of spinal cord injury and repair.

Spinal cord injury (SCI) lesions present diverse challenges for repair strategies. Anatomically complete injuries require restoration of neural connectivity across lesions. Anatomically incomplete injuries may benefit from augmentation of spontaneous circuit reorganization. Here, we review SCI cell biology, which varies considerably across three different lesion-related tissue compartments: (a) non-neural lesion core, (b) astrocyte scar border, and (c) surrounding spared but reactive neural tissue. After SCI, axon growth and circuit reorganization are determined by neuron-cell-autonomous mechanisms and by interactions among neurons, glia, and immune and other cells. These interactions are shaped by both the presence and the absence of growth-modulating molecules, which vary markedly in different lesion compartments. The emerging understanding of how SCI cell biology differs across lesion compartments is fundamental to developing rationally targeted repair strategies.

Biodegradable scaffolds promote tissue remodeling and functional improvement in non-human primates with acute spinal cord injury.

Tissue loss significantly reduces the potential for functional recovery after spinal cord injury. We previously showed that implantation of porous scaffolds composed of a biodegradable and biocompatible block copolymer of Poly-lactic-co-glycolic acid and Poly-l-lysine improves functional recovery and reduces spinal cord tissue injury after spinal cord hemisection injury in rats. Here, we evaluated the safety and efficacy of porous scaffolds in non-human Old-World primates (Chlorocebus sabaeus) after a partial and complete lateral hemisection of the thoracic spinal cord. Detailed analyses of kinematics and muscle activity revealed that by twelve weeks after injury fully hemisected monkeys implanted with scaffolds exhibited significantly improved recovery of locomotion compared to non-implanted control animals. Twelve weeks after injury, histological analysis demonstrated that the spinal cords of monkeys with a hemisection injury implanted with scaffolds underwent appositional healing characterized by a significant increase in remodeled tissue in the region of the hemisection compared to non-implanted controls. The number of glial fibrillary acidic protein immunopositive astrocytes was diminished within the inner regions of the remodeled tissue layer in treated animals. Activated macrophage and microglia were present diffusely throughout the remodeled tissue and concentrated at the interface between the preserved spinal cord tissue and the remodeled tissue layer. Numerous unphosphorylated neurofilament H and neuronal growth associated protein positive fibers and myelin basic protein positive cells may indicate neural sprouting inside the remodeled tissue layer of treated monkeys. These results support the safety and efficacy of polymer scaffolds in a primate model of acute spinal cord injury. A device substantially similar to the device described here is the subject of an ongoing human clinical trial.

Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury.

Ependyma have been proposed as adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after spinal cord injury (SCI). This proposal was based on small, midline stab SCI. Here, we tested the generality of this proposal by using a genetic knock-in cell fate mapping strategy in different murine SCI models. After large crush injuries across the entire spinal cord, ependyma-derived progeny remained local, did not migrate and contributed few cells of any kind and less than 2%, if any, of the total newly proliferated and molecularly confirmed scar-forming astrocytes. Stab injuries that were near to but did not directly damage ependyma, contained no ependyma-derived cells. Our findings show that ependymal contribution of progeny after SCI is minimal, local and dependent on direct ependymal injury, indicating that ependyma are not a major source of endogenous neural stem cells or neuroprotective astrocytes after SCI.

Synthesis and characterization of a library of in-situ curing, nonswelling ethoxylated polyol thiol-ene hydrogels for tailorable macromolecule delivery.

A transesterfication reaction is used to synthesize tri-thiol-functionalized-ethoxylated polyols that are combined with polyethylene glycol diacrylates to form a biodegradable hydrogel library. Hydrogels display nonswelling equilibration and offer temporal control over material degradation and the release of biomolecules. The demonstrated in vitro biocompatibility makes this a versatile platform that can be used for local drug delivery to volume-constrained anatomical sites.

Covalent Incorporation of Trehalose within Hydrogels for Enhanced Long-Term Functional Stability and Controlled Release of Biomacromolecules.

Hydrogels with covalently incorporated trehalose are synthesized using thiol-ene Michael addition. Trehalose hydrogels afford prolonged stabilization and -controlled release of model enzymes in vitro and in vivo as well as preservation of protein stability under heat and -lyophilization stressors. Strong and -ordered hydrogen bonding interactions within covalently incorporated trehalose hydrogels represent a possible mechanism for protein stabilization.

Evaluation of viscoelastic poly(ethylene glycol) sols as vitreous substitutes in an experimental vitrectomy model in rabbits.

The aim of this study was to employ an experimental protocol for in vivo evaluation of sols of 5 wt.% poly(ethylene glycol) (PEG) in phosphate-buffered saline as artificial vitreous substitutes. A 20 gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of eight pigmented rabbits. Approximately 1 ml of the viscoelastic PEG sols was then injected into the vitreous space of six eyes. PEG with an average molecular weight of 300,000 and 400,000 g mol(-1) was used in two and four eyes, respectively. Two eyes received balanced salt solution and served as controls. Full-field electroretinography was carried out and intra-ocular pressure (IOP, palpation) measured pre- and post-operatively at regular intervals up to 41 days. The rabbits were killed and the eyes examined by retinal photography, gross macroscopic examination and histology. The viscoelastic sols were successfully injected and remained translucent throughout the post-operative period, with some inferior formation of precipitates. None of the eyes displayed IOP elevation post-operatively, but in three of the PEG sol injected eyes transient hypotony was noted. One eye sustained retinal detachment during surgery and another two in the post-operative period. ERG recordings confirmed preservation of retinal function in three out of four eyes injected with 400,000 g mol(-1) PEG. Histological examination revealed up-regulation of glial acidic fibrillary protein in Müller cells in PEG sol injected eyes, but normal overall morphology in eyes with attached retinas. The viscosity of the sol was not retained throughout the post-operative period, indicating the demand for polymer cross-linking to increase residence time. The results provide promising preliminary results on the use of PEG hydrogels as a vitreous substitute.

An injectable thiol-acrylate poly(ethylene glycol) hydrogel for sustained release of methylprednisolone sodium succinate.

Clinically available injectable hydrogels face technical challenges associated with swelling after injection and toxicity from unreacted constituents that impede their performance as surgical biomaterials. To overcome these challenges, we developed a system where chemical gelation was controlled by a conjugate Michael addition between thiol and acrylate in aqueous media, with 97% monomer conversion and 6 wt.% sol fraction. The hydrogel exhibited syneresis on equilibration, reducing to 59.7% of its initial volume. It had mechanical properties similar to soft human tissue with an elastic modulus of 189.8 kPa. Furthermore, a mesh size of 6.9 nm resulted in sustained release of methylprednisolone sodium succinate with a loading efficiency of 2 mg/mL. Functionalization with 50 µg/mL of an oligolysine peptide resulted in attachment of freshly isolated murine mesenchymal stem cells. The rational design of the physical, chemical and biological properties of the hydrogel makes it a potentially promising candidate for injectable applications.

Bilayered scaffolds for osteochondral tissue engineering.

Osteoarthritis (OA) is a prevalent degenerative joint disease that places a significant burden on the socioeconomic efficacy of communities around the world. Tissue engineering repair of articular cartilage in synovial joints represents a potential OA treatment strategy superior to current surgical techniques. In particular, osteochondral tissue engineering, which promotes the simultaneous regeneration of articular cartilage and underlining subchondral bone, may be a clinically relevant approach toward impeding OA progression. The unique and complex functional demands of the two contrasting tissues that comprise osteochondral tissue require the use of bilayered scaffolds to promote individual growth of both on a single integrated implant. This paper reviews the three current bilayered scaffold strategies applied to solve this challenging problem, with a focus on the need for an innovative approach to design and fabrication of new optimized scaffold combinations to reinforce materials science as an important element of osteochondral tissue engineering.