Inhibition of Apoptosis in a Model of Ischemic Stroke Leads to Enhanced Cell Survival, Endogenous Neural Precursor Cell Activation and Improved Functional Outcomes.

Stroke results in neuronal cell death, which causes long-term disabilities in adults. Treatment options are limited and rely on a narrow window of opportunity. Apoptosis inhibitors demonstrate efficacy in improving neuronal cell survival in animal models of stroke. However, many inhibitors non-specifically target apoptosis pathways and high doses are needed for treatment. We explored the use of a novel caspase-3/7 inhibitor, New World Laboratories (NWL) 283, with a lower IC50 than current caspase-3/7 inhibitors. We performed in vitro and in vivo assays to determine the efficacy of NWL283 in modulating cell death in a preclinical model of stroke. In vitro and in vivo assays show that NWL283 enhances cell survival of neural precursor cells. Delivery of NWL283 following stroke enhances endogenous NPC migration and leads to increased neurogenesis in the stroke-injured cortex. Furthermore, acute NWL283 administration is neuroprotective at the stroke injury site, decreasing neuronal cell death and reducing microglia activation. Coincident with NWL283 delivery for 8 days, stroke-injured mice exhibited improved functional outcomes that persisted following cessation of the drug. Therefore, we propose that NWL283 is a promising therapeutic warranting further investigation to enhance stroke recovery.

Suppressing CSPG/LAR/PTPσ Axis Facilitates Neuronal Replacement and Synaptogenesis by Human Neural Precursor Grafts and Improves Recovery after Spinal Cord Injury.

Traumatic spinal cord injury (SCI) is a leading cause of permanent neurologic disabilities in young adults. Functional impairments after SCI are substantially attributed to the progressive neurodegeneration. However, regeneration of spinal-specific neurons and circuit re-assembly remain challenging in the dysregulated milieu of SCI because of impaired neurogenesis and neuronal maturation by neural precursor cells (NPCs) spontaneously or in cell-based strategies. The extrinsic mechanisms that regulate neuronal differentiation and synaptogenesis in SCI are poorly understood. Here, we perform extensive in vitro and in vivo studies to unravel that SCI-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) impedes neurogenesis of NPCs through co-activation of two receptor protein tyrosine phosphatases, LAR and PTPσ. In adult female rats with SCI, systemic co-inhibition of LAR and PTPσ promotes regeneration of motoneurons and spinal interneurons by engrafted human directly reprogramed caudalized NPCs (drNPC-O2) and fosters their morphologic maturity and synaptic connectivity within the host neural network that culminate in improved recovery of locomotion and sensorimotor integration. Our transcriptomic analysis of engrafted human NPCs in the injured spinal cord confirmed that inhibition of CSPG receptors activates a comprehensive program of gene expression in NPCs that can support neuronal differentiation, maturation, morphologic complexity, signal transmission, synaptic plasticity, and behavioral improvement after SCI. We uncovered that CSPG/LAR/PTPσ axis suppresses neuronal differentiation in part by blocking Wnt/ß-Catenin pathway. Taken together, we provide the first evidence that CSPGs/LAR/PTPσ axis restricts neurogenesis and synaptic integration of new neurons in NPC cellular therapies for SCI. We propose targeting LAR and PTPσ receptors offers a promising clinically-feasible adjunct treatment to optimize the efficacy and neurologic benefits of ongoing NPC-based clinical trials for SCI.SIGNIFICANCE STATEMENT Transplantation of neural precursor cells (NPCs) is a promising approach for replacing damaged neurons after spinal cord injury (SCI). However, survival, neuronal differentiation, and synaptic connectivity of transplanted NPCs within remain challenging in SCI. Here, we unravel that activation of chondroitin sulfate proteoglycan (CSPG)/LAR/PTPσ axis after SCI impedes the capacity of transplanted human NPCs for replacing functionally integrated neurons. Co-blockade of LAR and PTPσ is sufficient to promote re-generation of motoneurons and spinal V1 and V3 interneurons by engrafted human caudalized directly reprogramed NPCs (drNPC-O2) and facilitate their synaptic integration within the injured spinal cord. CSPG/LAR/PTPσ axis appears to suppress neuronal differentiation of NPCs by inhibiting Wnt/ß-Catenin pathway. These findings identify targeting CSPG/LAR/PTPσ axis as a promising strategy for optimizing neuronal replacement, synaptic re-connectivity, and neurologic recovery in NPC-based strategies.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates.

BACKGROUND: The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI. AIM: To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs). METHODS: Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle. RESULTS: Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones. CONCLUSION: Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.

Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action.

Cell therapy is an emerging approach to stroke treatment with a potential to limit brain damage and enhance its restoration after the acute phase of the disease. In this study we tested directly reprogrammed neural precursor cells (drNPC) derived from adult human bone marrow cells in the rat middle cerebral artery occlusion (MCAO) model of acute ischemic stroke using human placenta mesenchymal stem cells (pMSC) as a positive control with previously confirmed efficacy. Cells were infused into the ipsilateral (right) internal carotid artery of male Wistar rats 24 h after MCAO. The main goal of this work was to evaluate real-time distribution and subsequent homing of transplanted cells in the brain. This was achieved by performing intra-arterial infusion directly inside the MRI scanner and allowed transplanted cells tracing starting from their first pass through the brain vessels. Immediately after transplantation, cells were observed in the periphery of the infarct zone and in the brain stem, 15 min later small numbers of cells could be discovered deep in the infarct core and in the contralateral hemisphere, where drNPC were seen earlier and in greater numbers than pMSC. Transplanted cells in both groups could no longer be detected in the rat brain 48-72 h after infusion. Histological and histochemical analysis demonstrated that both the drNPC and pMSC were localized inside blood vessels in close contact with the vascular wall. No passage of labeled cells through the blood brain barrier was observed. Additionally, the therapeutic effects of drNPC and pMSC were compared. Both drNPC and pMSC induced substantial attenuation of neurological deficits evaluated at the 7th and 14th day after transplantation using the modified neurological severity score (mNSS). Some of the effects of drNPC and pMSC, such as the influence on the infarct volume and the survival rate of animals, differed. The results suggest a paracrine mechanism of the positive therapeutic effects of IA drNPC and pMSC infusion, potentially enhanced by the cell-cell interactions. Our data also indicate that the long-term homing of transplanted cells in the brain is not necessary for the brain's functional recovery.

Substrate-Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells.

Background: Neural precursor cells (NPCs) hold great promise for neural repair. Endogenous NPCs, found in the subventricular zone of the adult brain, proliferate and migrate toward lesion sites; however, it is not sufficient for neural repair. NPCs are electrosensitive cells that undergo directed migration in an electric field (EF). Here, we examined the EF-induced migration of a clinically relevant human NPC population. Materials & Methods: We examined the effects of different substrates and microenvironments on human NPC galvanotaxis. Results: Human NPCs increased their migration speed in the presence of an EF, and the direction of migration (anodal vs. cathodal) varied between substrates. The secretome and extracellular pH were not significant factors in EF-induced migration; however, our results are consistent with substrate stiffness playing a role in the direction of cell migration. Conclusion: These findings provide insight into the importance of the microenvironment on modulating human NPC migration and highlight substrate-dependent considerations for neurorepair.

Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

Stroke is one of the leading causes of long-term disability. Cell transplantation is a promising strategy to treat stroke. We explored the efficacy of directly reprogrammed human neural precursor cell (drNPC) transplants to promote functional recovery in a model of focal ischemic stroke in the mouse sensorimotor cortex. We show that drNPCs express neural precursor cell markers and are neurally committed at the time of transplantation. Mice that received drNPC transplants recovered motor function, irrespective of transplant vehicle or recipient sex, and with no correlation to lesion volume or glial scarring. The majority of drNPCs found in vivo, at the time of functional recovery, remained undifferentiated. Notably, no correlation between functional recovery and long-term xenograft survival was observed, indicating that drNPCs provide therapeutic benefits beyond their survival. Furthermore, increased synaptophysin expression in transplanted brains suggests that drNPCs promote neuroplasticity through enhanced synaptogenesis. Our findings provide insight into the mechanistic underpinnings of drNPC-mediated recovery for stroke and support the notion that drNPCs may have clinical applications for stroke therapy.

Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

BACKGROUND: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. METHODS: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. RESULTS: Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. CONCLUSIONS: Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

Human Oligodendrogenic Neural Progenitor Cells Delivered with Chondroitinase ABC Facilitate Functional Repair of Chronic Spinal Cord Injury.

Treatment of chronic spinal cord injury (SCI) is challenging due to cell loss, cyst formation, and the glial scar. Previously, we reported on the therapeutic potential of a neural progenitor cell (NPC) and chondroitinase ABC (ChABC) combinatorial therapy for chronic SCI. However, the source of NPCs and delivery system required for ChABC remained barriers to clinical application. Here, we investigated directly reprogrammed human NPCs biased toward an oligodendrogenic fate (oNPCs) in combination with sustained delivery of ChABC using an innovative affinity release strategy in a crosslinked methylcellulose biomaterial for the treatment of chronic SCI in an immunodeficient rat model. This combinatorial therapy increased long-term survival of oNPCs around the lesion epicenter, facilitated greater oligodendrocyte differentiation, remyelination of the spared axons by engrafted oNPCs, enhanced synaptic connectivity with anterior horn cells and neurobehavioral recovery. This combinatorial therapy is a promising strategy to regenerate the chronically injured spinal cord.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing.

Cell transplantation therapy utilizing neural precursor cells (NPCs) is a conceptually attractive strategy for traumatic spinal cord injury (SCI) to replace lost cells, remyelinate denuded host axons and promote tissue sparing. However, the number of mature oligodendrocytes that differentiate from typical NPCs remains limited. Herein, we describe a novel approach to bias the differentiation of directly reprogrammed human NPCs (drNPCs) toward a more oligodendrogenic fate (oNPCs) while preserving their tripotency. The oNPCs derived from different lines of human NPCs showed similar characteristics in vitro. To assess the in vivo efficacy of this approach, we used oNPCs derived from drNPCs and transplanted them into a SCI model in immunodeficient Rowett Nude (RNU) rats. The transplanted cells showed significant migration along the rostrocaudal axis and proportionally greater differentiation into oligodendrocytes. These cells promoted perilesional tissue sparing and axonal remyelination, which resulted in recovery of motor function. Moreover, after transplantation of the oNPCs into intact spinal cords of immunodeficient NOD/SCID mice, we detected no evidence of tumor formation even after 5 months of observation. Thus, biasing drNPC differentiation along an oligodendroglial lineage represents a promising approach to promote tissue sparing, axonal remyelination, and neural repair after traumatic SCI. Stem Cells Translational Medicine 2018;7:806-818.

Caspase vinyl sulfone small molecule inhibitors prevent axonal degeneration in human neurons and reverse cognitive impairment in Caspase-6-overexpressing mice.

BACKGROUND: The activation of the aspartate-specific cysteinyl protease, Caspase-6, is proposed as an early pathogenic event of Alzheimer disease (AD) and Huntington's disease. Caspase-6 inhibitors could be useful against these neurodegenerative diseases but most Caspase-6 inhibitors have been exclusively studied in vitro or show acute liver toxicity in humans. Here, we assessed vinyl sulfone small molecule peptide caspase inhibitors for potential use in vivo. METHODS: The IC50 of NWL vinyl sulfone small molecule caspase inhibitors were determined on Caspase-1 to 10, and Caspase-6-transfected human colon carcinoma HCT116 cells. Inhibition of Caspase-6-mediated axonal degeneration was assessed in serum-deprived or amyloid precursor protein-transfected primary human CNS neurons. Cellular toxicity was measured by phase contrast microscopy, mitochondrial and lactate dehydrogenase colorimetric activity assays, or flow cytometry. Caspase inhibition was measured by fluorogenic activity assays, fluorescence microscopy, and western blot analyses. The effect of inhibitors on age-dependent cognitive deficits in Caspase-6 transgenic mice was assessed by the novel object recognition task. Liquid chromatography coupled to tandem mass spectrometry assessed the blood-brain barrier permeability of inhibitors in Caspase-6 mice. RESULTS: Vinyl sulfone NWL-117 caspase inhibitor has a higher selectivity against Caspase-6, -4, -8, -9, and -10 whereas NWL-154 has higher selectivity against Caspase-6, -8, and -10. The half-maximal inhibitory concentrations (IC50) of NWL-117 and NWL-154 is 192 nM and 100 nM against Caspase-6 in vitro, and 4.82 µM and 3.63 µM in Caspase-6-transfected HCT116 cells, respectively. NWL inhibitors are not toxic to HCT116 cells or to human primary neurons. NWL-117 and NWL-154 inhibit serum deprivation-induced Caspase-6 activity and prevent amyloid precursor protein-mediated neurite degeneration in human primary CNS neurons. NWL-117 crosses the blood brain barrier and reverses age-dependent episodic memory deficits in Caspase-6 mice. CONCLUSIONS: NWL peptidic vinyl methyl sulfone inhibitors are potent, non-toxic, blood-brain barrier permeable, and irreversible caspase inhibitors with neuroprotective effects in HCT116 cells, in primary human CNS neurons, and in Caspase-6 mice. These results highlight the therapeutic potential of vinyl sulfone inhibitors as caspase inhibitors against neurodegenerative diseases and sanction additional work to improve their selectivity against different caspases.

Biomechanical and biochemical characteristics of a human fibroblast-produced and remodeled matrix.

We report on a culture method for the rapid production of a strong and thick natural matrix by human cells for tissue engineering applications. Dermal fibroblasts were cultured for three weeks at high density on porous substrates in serum-containing or chemically defined media. The mechanical and biochemical properties of the resulting cell-derived matrix (CDM) were compared to those of standard fibroblast-populated collagen and fibrin gels and native human skin. We found that the ultimate tensile strength of CDM cultured in our chemically defined media (313+/-8.7 kPa) is significantly greater than for collagen gels (168+/-39.3 kPa), fibrin gels (133+/-8.0 kPa) and CDM cultured with serum (223+/-9.0 kPa), but less than native skin (713+/-55.2 kPa). In addition to the biomechanics, this *CDM is also biochemically more similar to native skin than the collagen and fibrin gels in terms of all parameters measured. As *CDM is produced by human cells in a chemically defined culture medium and is mechanically robust, it may be a viable living tissue equivalent for many connective tissue replacement applications requiring initial mechanical stability yet a high degree of biocompatibility.