Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Inhibiting USP16 rescues stem cell aging and memory in an Alzheimer's model.

Alzheimer's disease (AD) is a progressive neurodegenerative disease observed with aging that represents the most common form of dementia. To date, therapies targeting end-stage disease plaques, tangles, or inflammation have limited efficacy. Therefore, we set out to identify a potential earlier targetable phenotype. Utilizing a mouse model of AD and human fetal cells harboring mutant amyloid precursor protein, we show cell intrinsic neural precursor cell (NPC) dysfunction precedes widespread inflammation and amyloid plaque pathology, making it the earliest defect in the evolution of the disease. We demonstrate that reversing impaired NPC self-renewal via genetic reduction of USP16, a histone modifier and critical physiological antagonist of the Polycomb Repressor Complex 1, can prevent downstream cognitive defects and decrease astrogliosis in vivo. Reduction of USP16 led to decreased expression of senescence gene Cdkn2a and mitigated aberrant regulation of the Bone Morphogenetic Signaling (BMP) pathway, a previously unknown function of USP16. Thus, we reveal USP16 as a novel target in an AD model that can both ameliorate the NPC defect and rescue memory and learning through its regulation of both Cdkn2a and BMP signaling.

Novel C1q receptor-mediated signaling controls neural stem cell behavior and neurorepair.

C1q plays a key role as a recognition molecule in the immune system, driving autocatalytic complement cascade activation and acting as an opsonin. We have previously reported a non-immune role of complement C1q modulating the migration and fate of human neural stem cells (hNSC); however, the mechanism underlying these effects has not yet been identified. Here, we show for the first time that C1q acts as a functional hNSC ligand, inducing intracellular signaling to control cell behavior. Using an unbiased screening strategy, we identified five transmembrane C1q signaling/receptor candidates in hNSC (CD44, GPR62, BAI1, c-MET, and ADCY5). We further investigated the interaction between C1q and CD44 , demonstrating that CD44 mediates C1q induced hNSC signaling and chemotaxis in vitro, and hNSC migration and functional repair in vivo after spinal cord injury. These results reveal a receptor-mediated mechanism for C1q modulation of NSC behavior and show that modification of C1q receptor expression can expand the therapeutic window for hNSC transplantation.

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells - Tools for analyzing dynamics of cell cycle, migration, and lineage selection.

Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7-14 day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7 days as well as lineage selection and migration using human NSC for 14 days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24-72 h up to 7-14 days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis.

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.

Increasing Human Neural Stem Cell Transplantation Dose Alters Oligodendroglial and Neuronal Differentiation after Spinal Cord Injury.

Multipotent human central nervous system-derived neural stem cells transplanted at doses ranging from 10,000 (low) to 500,000 (very high) cells differentiated predominantly into the oligodendroglial lineage. However, while the number of engrafted cells increased linearly in relationship to increasing dose, the proportion of oligodendrocytic cells declined. Increasing dose resulted in a plateau of engraftment, enhanced neuronal differentiation, and increased distal migration caudal to the transplantation sites. Dose had no effect on terminal sensory recovery or open-field locomotor scores. However, total human cell number and decreased oligodendroglial proportion were correlated with hindlimb girdle coupling errors. Conversely, greater oligodendroglial proportion was correlated with increased Ab step pattern, decreased swing speed, and increased paw intensity, consistent with improved recovery. These data suggest that transplant dose, and/or target niche parameters can regulate donor cell engraftment, differentiation/maturation, and lineage-specific migration profiles.

Preclinical Efficacy Failure of Human CNS-Derived Stem Cells for Use in the Pathway Study of Cervical Spinal Cord Injury.

We previously showed the efficacy of multiple research cell lines (RCLs) of human CNS neural stem cells (HuCNS-SCs) in mouse and rat models of thoracic spinal cord injury (SCI), supporting a thoracic SCI clinical trial. Experts recommend in vivo preclinical testing of the intended clinical cell lot/line (CCL) in models with validity for the planned clinical target. We therefore tested the efficacy of two HuCNS-SC lines in cervical SCI: one RCL, and one CCL intended for use in the Pathway Study of cervical SCI in man. We assessed locomotor recovery and sensory function, as well as engraftment, migration, and fate. No evidence of efficacy of the CCL was observed; some data suggested a negative impact of the CCL on outcomes. These data raise questions about the development and validation of potency/comparability assays for clinical testing of cell products, and lack of US Food and Drug Administration requirements for in vivo testing of intended clinical cell lines.

Transplantation dose alters the dynamics of human neural stem cell engraftment, proliferation and migration after spinal cord injury.

The effect of transplantation dose on the spatiotemporal dynamics of human neural stem cell (hNSC) engraftment has not been quantitatively evaluated in the central nervous system. We investigated changes over time in engraftment/survival, proliferation, and migration of multipotent human central nervous system-derived neural stem cells (hCNS-SCns) transplanted at doses ranging from 10,000 to 500,000 cells in spinal cord injured immunodeficient mice. Transplant dose was inversely correlated with measures of donor cell proliferation at 2 weeks post-transplant (WPT) and dose-normalized engraftment at 16 WPT. Critically, mice receiving the highest cell dose exhibited an engraftment plateau, in which the total number of engrafted human cells never exceeded the initial dose. These data suggest that donor cell expansion was inversely regulated by target niche parameters and/or transplantation density. Investigation of the response of donor cells to the host microenvironment should be a key variable in defining target cell dose in pre-clinical models of CNS disease and injury.

Safety of human neural stem cell transplantation in chronic spinal cord injury.

The spinal cord injury (SCI) microenvironment undergoes dynamic changes over time, which could potentially affect survival or differentiation of cells in early versus delayed transplantation study designs. Accordingly, assessment of safety parameters, including cell survival, migration, fate, sensory fiber sprouting, and behavioral measures of pain sensitivity in animals receiving transplants during the chronic postinjury period is required for establishing a potential therapeutic window. The goal of the study was assessment of safety parameters for delayed transplantation of human central nervous system-derived neural stem cells (hCNS-SCns) by comparing hCNS-SCns transplantation in the subacute period, 9 days postinjury (DPI), versus the chronic period, 60 DPI, in contusion-injured athymic nude rats. Although the number of surviving human cells after chronic transplantation was lower, no changes in cell migration were detected between the 9 and 60 DPI cohorts; however, the data suggest chronic transplantation may have enhanced the generation of mature oligodendrocytes. The timing of transplantation did not induce changes in allodynia or hyperalgesia measures. Together, these data support the safety of hCNS-SCns transplantation in the chronic period post-SCI.

Safety of epicenter versus intact parenchyma as a transplantation site for human neural stem cells for spinal cord injury therapy.

Neural stem cell transplantation may have the potential to yield repair and recovery of function in central nervous system injury and disease, including spinal cord injury (SCI). Multiple pathological processes are initiated at the epicenter of a traumatic spinal cord injury; these are generally thought to make the epicenter a particularly hostile microenvironment. Conversely, the injury epicenter is an appealing potential site of therapeutic human central nervous system-derived neural stem cell (hCNS-SCns) transplantation because of both its surgical accessibility and the avoidance of spared spinal cord tissue. In this study, we compared hCNS-SCns transplantation into the SCI epicenter (EPI) versus intact rostral/caudal (R/C) parenchyma in contusion-injured athymic nude rats, and assessed the cell survival, differentiation, and migration. Regardless of transplantation site, hCNS-SCns survived and proliferated; however, the total number of hCNS-SCns quantified in the R/C transplant animals was twice that in the EPI animals, demonstrating increased overall engraftment. Migration and fate profile were unaffected by transplantation site. However, although transplantation site did not alter the proportion of human astrocytes, EPI transplantation shifted the localization of these cells and exhibited a correlation with calcitonin gene-related peptide fiber sprouting. Critically, no changes in mechanical allodynia or thermal hyperalgesia were observed. Taken together, these data suggest that the intact parenchyma may be a more favorable transplantation site than the injury epicenter in the subacute period post-SCI.

Computer-aided 2D and 3D quantification of human stem cell fate from in vitro samples using Volocity high performance image analysis software.

Accurate automated cell fate analysis of immunostained human stem cells from 2- and 3-dimensional (2D-3D) images would improve efficiency in the field of stem cell research. Development of an accurate and precise tool that reduces variability and the time needed for human stem cell fate analysis will improve productivity and interpretability of the data across research groups. In this study, we have created protocols for high performance image analysis software Volocity® to classify and quantify cytoplasmic and nuclear cell fate markers from 2D-3D images of human neural stem cells after in vitro differentiation. To enhance 3D image capture efficiency, we optimized the image acquisition settings of an Olympus FV10i® confocal laser scanning microscope to match our quantification protocols and improve cell fate classification. The methods developed in this study will allow for a more time efficient and accurate software based, operator validated, stem cell fate classification and quantification from 2D and 3D images, and yield the highest ≥94.4% correspondence with human recognized objects.

Myc increases self-renewal in neural progenitor cells through Miz-1.

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process, we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold, and 1% of the Myc-overexpressing cells, but none of the control cells, retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1, did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc, indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc, through Miz-1, enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells.

Characterization of the NF2 protein merlin and the ERM protein ezrin in human, rat, and mouse central nervous system.

The neurofibromatosis 2 (NF2) protein, merlin, is structurally related to the ERM (ezrin-radixin-moesin) protein family of membrane-cytoskeleton linkers and is mutated in nervous system tumors. Apart from tumor suppressor activity, merlin's functions are poorly understood. We compared the localization and expression of merlin and ezrin in developing and adult brain and in brain-derived progenitor cells. Both proteins were widely but differentially expressed in human, rat, and mouse brain. In brain tissue and neuronal progenitor cell cultures merlin was predominantly found in neurons while ezrin was expressed in astrocytes. Merlin expression was seen from E11 in mouse embryos, whereas ezrin was present earlier. Both proteins were expressed in embryonic mouse neurospheres, where ezrin was specifically localized in filopodia of adherent neuronal progenitor cells. Subcellular analysis demonstrated ezrin in fine filopodial structures in astrocytes, while merlin was detected in neuronal synaptic junctions. The widespread expression of merlin in brain and its association with protein kinase A suggest a role for merlin in brain biology.

Surgical recovery and successful surgical transfer of conventionally frozen-thawed embryos in the farmed European polecat (Mustela putorius).

Surgical transfer of in vivo produced conventionally frozen-thawed embryos of farmed European polecat (Mustela putorius) was investigated as a part of an ex-situ preservation program which has the long-term aim of developing a genome resource bank for the endangered European mink (Mustela lutreola). Eighteen oestrous yearling European polecat donors were mated once daily on two consecutive days using 13 fertile males. The donors were surgically flushed for embryos 8-9 days after the first mating. The embryo recovery rate was 60% (116 embryos/193 corpora lutea). The embryos were cryopreserved with 1.5 M ethylene glycol in a programmable freezer using a conventional slow freezing protocol. The thawed embryos were surgically transferred either after dilution with 0.5 M sucrose or directly without removal of ethylene glycol. To induce ovulation, eight recipient females were mated once daily on two consecutive days with vasectomized males starting 7 or 8 days before embryo transfer. The recipients received 7-11 embryos each and three recipients delivered a total of nine pups after a gestation length of 44-46 days. The embryo survival rate was 10% (9 pups/93 frozen embryos). This report describes the first successful cryopreservation of embryos in the Mustelidae family resulting in viable offspring. The low embryo survival rate, however, indicates that the freezing-thawing protocol needs to be improved.