Ectopic Expression of Neurod1 Is Sufficient for Functional Recovery following a Sensory-Motor Cortical Stroke.

Stroke is the leading cause of adult disability worldwide. The majority of stroke survivors are left with devastating functional impairments for which few treatment options exist. Recently, a number of studies have used ectopic expression of transcription factors that direct neuronal cell fate with the intention of converting astrocytes to neurons in various models of brain injury and disease. While there have been reports that question whether astrocyte-to-neuron conversion occurs in vivo, here, we have asked if ectopic expression of the transcription factor Neurod1 is sufficient to promote improved functional outcomes when delivered in the subacute phase following endothelin-1-induced sensory-motor cortex stroke. We used an adeno-associated virus to deliver Neurod1 from the short GFAP promoter and demonstrated improved functional outcomes as early as 28 days post-stroke and persisting to at least 63 days post-stroke. Using Cre-based cell fate tracking, we showed that functional recovery correlated with the expression of neuronal markers in transduced cells by 28 days post-stroke. By 63 days post-stroke, the reporter-expressing cells comprised ~20% of all the neurons in the perilesional cortex and expressed markers of cortical neuron subtypes. Overall, our findings indicate that ectopic expression of Neurod1 in the stroke-injured brain is sufficient to enhance neural repair.

Constraint-induced movement therapy promotes motor recovery after neonatal stroke in the absence of neural precursor activation.

Neonatal stroke is a leading cause of long-term disability and currently available rehabilitation treatments are insufficient to promote recovery. Activating neural precursor cells (NPCs) in adult rodents, in combination with rehabilitation, can accelerate functional recovery following stroke. Here, we describe a novel method of constraint-induced movement therapy (CIMT) in a rodent model of neonatal stroke that leads to improved functional outcomes, and we asked whether the recovery was correlated with expansion of NPCs. A hypoxia/ischemia (H/I) injury was induced on postnatal day 8 (PND8) via unilateral carotid artery ligation followed by systemic hypoxia. One week and two weeks post-H/I, CIMT was administered in the form of 3 botulinum toxin (Botox) injections, which induced temporary paralysis in the unaffected limb. Functional recovery was assessed using the foot fault task. NPC proliferation was assessed using the neurosphere assay and EdU immunohistochemistry. We found that neonatal H/I injury alone expands the NPC pool by >2.5-fold relative to controls. We determined that using Botox injections as a method to provide CIMT results in significant functional motor recovery after H/I. However, CIMT does not lead to enhanced NPC activation or migration into the injured parenchyma in vivo. At the time of functional recovery, increased numbers of proliferating inflammatory cells were found within the injured motor cortex. Together, these findings suggest that NPC activation following CIMT does not account for the observed functional improvement and suggests that CIMT-mediated modification of the CNS inflammatory response may play a role in the motor recovery.

Lineage tracing reveals the hierarchical relationship between neural stem cell populations in the mouse forebrain.

Since the original isolation of neural stem cells (NSCs) in the adult mammalian brain, further work has revealed a heterogeneity in the NSC pool. Our previous work characterized a distinct, Oct4 expressing, NSC population in the periventricular region, through development and into adulthood. We hypothesized that this population is upstream in lineage to the more abundant, well documented, GFAP expressing NSC. Herein, we show that Oct4 expressing NSCs give rise to neurons, astrocytes and oligodendrocytes throughout the developing brain. Further, transgenic inducible mouse models demonstrate that the rare Oct4 expressing NSCs undergo asymmetric divisions to give rise to GFAP expressing NSCs in naïve and injured brains. This lineage relationship between distinct NSC pools contributes significantly to an understanding of neural development, the NSC lineage in vivo and has implications for neural repair.

Myelin Basic Protein Regulates Primitive and Definitive Neural Stem Cell Proliferation from the Adult Spinal Cord.

The adult mammalian forebrain comprises two distinct populations of neural stem cells (NSCs): rare, Oct4 positive, primitive NSCs (pNSCs) and definitive NSC (dNSC) which are more abundant and express GFAP. The pNSCs are upstream of the dNSCs in the neural stem cell lineage. Herein we show that pNSC and dNSC populations can also be isolated from the developing and adult spinal cord. Spinal cord derived pNSCs are similarly rare, Oct4 expressing cells that are responsive to leukemia inhibitory factor and dNSCs are 4-5X more abundant and express GFAP. We demonstrate that myelin basic protein (MBP) is inhibitory to both pNSC and dNSC derived colony formation. Similar to what is seen in the adult forebrain following injury, spinal cord injury results in a significant increase in the size of the dNSC and pNSC pools. Hence, both primitive and definitive neural stem cells can be isolated from along the embryonic and adult neuraxis in vivo and their behavior is regulated by MBP and injury. Stem Cells 2017;35:485-496.

Adult Neural Stem Cells from the Subventricular Zone Give Rise to Reactive Astrocytes in the Cortex after Stroke.

Reactive astrocytes (RAs) have been reported to convert to multipotent neural stem cells (NSCs) capable of neurosphere (NS) formation and multilineage differentiation in vitro. Using genetic tagging, we determined that subventricular zone (SVZ) NSCs give rise to NSs derived from the stroke-injured cortex. We demonstrate that these cells can be isolated from the cortex in two different models of stroke and from different stroke-lesioned cortical regions. Interestingly, SVZ NSCs give rise to a subpopulation of RAs in the cortex that contribute to astrogliosis and scar formation. Last, we show that these SVZ derived RAs can be converted to neurons in vivo by forced expression of Ascl1. Identifying the contribution of cells originating from the SVZ to injury repair has implications for neural regeneration strategies.

Cyclosporin A enhances neural precursor cell survival in mice through a calcineurin-independent pathway.

Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neural precursor cells; NPCs) in the adult central nervous system. Administration of CsA in vitro or in vivo promotes the survival of NPCs and expands the pools of NPCs in mice. Moreover, CsA administration is effective in promoting NPC activation, tissue repair and functional recovery in a mouse model of cortical stroke. The mechanism(s) by which CsA mediates this cell survival effect remains unknown. Herein, we examined both calcineurin-dependent and calcineurin-independent pathways through which CsA might mediate NPC survival. To examine calcineurin-dependent pathways, we utilized FK506 (Tacrolimus), an immunosuppressive molecule that inhibits calcineurin, as well as drugs that inhibit cyclophilin A-mediated activation of calcineurin. To evaluate the calcineurin-independent pathway, we utilized NIM811, a non-immunosuppressive CsA analog that functions independently of calcineurin by blocking mitochondrial permeability transition pore formation. We found that only NIM811 can entirely account for the pro-survival effects of CsA on NPCs. Indeed, blocking signaling pathways downstream of calcineurin activation using nNOS mice did not inhibit CsA-mediated cell survival, which supports the proposal that the effects are calcinuerin-independent. In vivo studies revealed that NIM811 administration mimics the pro-survival effects of CsA on NPCs and promotes functional recovery in a model of cortical stroke, identical to the effects seen with CsA administration. We conclude that CsA mediates its effect on NPC survival through calcineurin-independent inhibition of mitochondrial permeability transition pore formation and suggest that this pathway has potential therapeutic benefits for developing NPC-mediated cell replacement strategies.

Primitive neural stem cells in the adult mammalian brain give rise to GFAP-expressing neural stem cells.

Adult forebrain definitive neural stem cells (NSCs) comprise a subpopulation of GFAP-expressing subependymal cells that arise from embryonic fibroblast growth factor (FGF)-dependent NSCs that are first isolated from the developing brain at E8.5. Embryonic FGF-dependent NSCs are derived from leukemia inhibitory factor (LIF)-responsive, Oct4-expressing primitive NSCs (pNSCs) that are first isolated at E5.5. We report the presence of a rare population of pNCSs in the periventricular region of the adult forebrain. Adult-derived pNSCs (AdpNSCs) are GFAP(-), LIF-responsive stem cells that display pNSC properties, including Oct4 expression and the ability to integrate into the inner cell mass of blastocysts. AdpNSCs generate self-renewing, multipotent colonies that give rise to definitive GFAP(+) NSCs in vitro and repopulate the subependyma after the ablation of GFAP(+) NSCs in vivo. These data support the hypothesis that a rare population of pNSCs is present in the adult brain and is upstream of the GFAP(+) NSCs.

Prosurvival factors derived from the embryonic brain promote adult neural stem cell survival.

Temporally distinct populations of neural stem cells (NSCs; embryonic and adult) display the cardinal stem cell properties of self-renewal and multipotentiality; however, their relative frequency and cell kinetics vary through development and into old age. We asked whether changes in NSC behavior could be accounted for by changes in environmental signals over time. We identified a prosurvival signaling cascade that enhances adult-derived NSC survival using cues released from embryonic neurons. Specifically, we demonstrate that stromal-cell-derived factor-1α (SDF-1α) released by embryonic neurons leads to upregulation of neuronal nitric oxide synthase in adult neural precursor cells. The resulting increase in nitric oxide leads to the upregulation of the stem cell factor (SCF) receptor ckit on adult NSCs (ANSCs). SCF released from embryonic neurons results in enhanced NSC survival. Using both in vitro and in vivo assays, we have demonstrated expansion of the size of the NSC pool through this pathway, indicating that ANSCs retain their ability to respond to embryonic-derived cues into adulthood.

Transient maternal IL-6 mediates long-lasting changes in neural stem cell pools by deregulating an endogenous self-renewal pathway.

The mechanisms that regulate the establishment of adult stem cell pools during normal and perturbed mammalian development are still largely unknown. Here, we asked whether a maternal cytokine surge, which occurs during human maternal infections and has been implicated in cognitive disorders, might have long-lasting consequences for neural stem cell pools in adult progeny. We show that transient, maternally administered interleukin-6 (IL-6) resulted in an expanded adult forebrain neural precursor pool and perturbed olfactory neurogenesis in offspring months after fetal exposure. This increase is likely the long-term consequence of acute hyperactivation of an endogenous autocrine/paracrine IL-6-dependent self-renewal pathway that normally regulates the number of forebrain neural precursors. These studies therefore identify an IL-6-dependent neural stem cell self-renewal pathway in vivo, and support a model in which transiently increased maternal cytokines can act through this pathway in offspring to deregulate neural precursor biology from embryogenesis throughout life.

Bioengineered sequential growth factor delivery stimulates brain tissue regeneration after stroke.

Stroke is a leading cause of disability with no effective regenerative treatment. One promising strategy for achieving tissue repair involves the stimulation of endogenous neural stem/progenitor cells through sequential delivery of epidermal growth factor (EGF) followed by erythropoietin (EPO). Yet currently available delivery strategies such as intracerebroventricular (ICV) infusion cause significant tissue damage. We designed a novel delivery system that circumvents the blood brain barrier and directly releases growth factors to the brain. Sequential release of the two growth factors is a key in eliciting tissue repair. To control release, we encapsulate pegylated EGF (EGF-PEG) in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and EPO in biphasic microparticles comprised of a PLGA core and a poly(sebacic acid) coating. EGF-PEG and EPO polymeric particles are dispersed in a hyaluronan methylcellulose (HAMC) hydrogel which spatially confines the particles and attenuates the inflammatory response of brain tissue. Our composite-mediated, sequential delivery of EGF-PEG and EPO leads to tissue repair in a mouse stroke model and minimizes damage compared to ICV infusion.

Transport of epidermal growth factor in the stroke-injured brain.

Stroke is a neurological disorder that currently has no cure. Intrathecal delivery of growth factors, specifically recombinant human epidermal growth factor (rhEGF), stimulates endogenous neural precursor cells in the subventricular zone (SVZ) and promotes tissue regeneration in animal models of stroke. In this model, rhEGF is delivered with an invasive minipump/catheter system, which causes trauma to the brain. A less invasive strategy is to deliver rhEGF from the brain cortex; however, this requires the protein to diffuse through the brain, from the site of injection to the SVZ. Although this method of delivery has great potential, diffusion is limited by rapid removal from the extracellular space and hence for successful translation into the clinic strategies are needed to increase the diffusion distance. Using integrative optical imaging we investigate diffusion of rhEGF vs. poly(ethylene glycol)-modified rhEGF (PEG-rhEGF) in brain slices of both uninjured and stroke-injured animals. For the first time, we quantitatively show that PEG modification reduces the rate of growth factor elimination by over an order of magnitude. For rhEGF this corresponds to a two to threefold increase in predicted brain penetration distance, which we confirm with in vivo data.

Neurogenic potential of isolated precursor cells from early post-gastrula somitic tissue.

Adult tissues are known to contain rare populations of stem cells with multilineage differentiation potential that are distinct from other resident tissue-specific stem cells. However, whether multilineage stem cells are involved in tissue development is uncertain, primarily because the identification and characterization of these cells in embryonic tissue primordia is not well established. We tested whether stem cells with multilineage potential are present within the early post-gastrula somite tissue. We show that clonally derived precursor cells generate colonies with self-renewal capacity and have both neurogenic and myogenic lineage potential. Somite colonies contain cells that express Sox2, nestin, and Sca1, but do not express genes indicative of somitic mesoderm specification. Furthermore, we demonstrate that this multilineage potential is not due to colony cells with a pluripotent epiblast identity or the selection of p75 receptor-positive neural crest stem cells. Despite utilizing a highly undifferentiated tissue source, colony formation was not enhanced relative to reported estimates of multilineage stem cells from adult muscle, a derivative of the embryonic somite. Thus, our findings suggest that a permissive in vitro environment is sufficient for the isolation of a discrete population of stem cells in the embryonic somite that may represent the earliest developmental precursor to adult muscle multilineage stem cells.