C. Morshead - Academia.edu (original) (raw)
Papers by C. Morshead
Journal of Magnetic Resonance Imaging, 2013
To evaluate the feasibility of using micron-sized superparamagnetic iron oxide particles (MPIOs) ... more To evaluate the feasibility of using micron-sized superparamagnetic iron oxide particles (MPIOs) as an effective labeling agent for monitoring bone marrow-derived mesenchymal stromal cell (BMSC) migration in the brain using magnetic resonance imaging (MRI) in a rat model of stroke and whether the accumulation of MPIO-labeled BMSCs can be differentiated from the accumulation of free MPIO particles or hemoglobin breakdown at a site of neuronal damage. In this study BMSCs were labeled with iron oxide and their pattern of migration following intravenous injection in a rat stroke model was monitored using a clinical MRI system followed by standard histopathology. The migration pattern was compared between intravenous injection of BMSCs alone, BMSCs labeled with MPIOs, and MPIO particles alone. The results demonstrated that while MRI was highly sensitive in the detection of iron oxide particle-containing cells in areas of neuronal ischemia, the true origin of cells containing iron oxide particles remains ambiguous. Therefore, detection of iron particles may not be a suitable strategy for the detection of BMSCs in the brain in a stroke model. This study suggests that the use of MPIOs as labeling agents are insufficient to conclusively determine the localization of iron within cells in regions of neuronal ischemia and hemorrhage.
Journal of Cerebral Blood Flow & Metabolism, 2006
Recent studies suggest that proliferation in the adult forebrain subventricular zone increases in... more Recent studies suggest that proliferation in the adult forebrain subventricular zone increases in response to a forebrain stroke and intraventricular infusions of growth factors enhance this response. The potential for growth factor infusions to regenerate the damaged motor cortex and promote recovery of motor function after stroke has not been examined. Here, we report that intraventricular infusions of epidermal growth factor and erythropoietin together, but not individually, promote substantial regeneration of the damaged cerebral cortex and reverse impairments in spontaneous and skilled motor tasks, in a rat model of stroke. Cortical regeneration and functional recovery occurred even when growth factor administration was delayed for up to 7 days after the stroke-induced lesion. Cell tracking demonstrated the contribution of neural precursors originating in the forebrain subventricular zone to the regenerated cortex. Strikingly, removal of the regenerated cortical tissue reversed...
Acta Pharmacologica Sinica, 2012
Stem cell-based therapies for the treatment of stroke have received considerable attention. Two b... more Stem cell-based therapies for the treatment of stroke have received considerable attention. Two broad approaches to stem cell-based therapies have been taken: the transplantation of exogenous stem cells, and the activation of endogenous neural stem and progenitor cells (together termed neural precursors). Studies examining the transplantation of exogenous cells have demonstrated that neural stem and progenitor cells lead to the most clinically promising results. Endogenous activation of neural precursors has also been explored based on the fact that resident precursor cells have the inherent capacity to proliferate, migrate and differentiate into mature neurons in the uninjured adult brain. Studies have revealed that these neural precursor cell behaviours can be activated following stroke, whereby neural precursors will expand in number, migrate to the infarct site and differentiate into neurons. However, this innate response is insufficient to lead to functional recovery, making it necessary to enhance the activation of endogenous precursors to promote tissue repair and functional recovery. Herein we will discuss the current state of the stem cell-based approaches with a focus on endogenous repair to treat the stroke injured brain.
Introduction: In animal models of stroke, endogenous neural precursor cells can be activated with... more Introduction: In animal models of stroke, endogenous neural precursor cells can be activated with growth factors such as epidermal growth factor (EGF) and erythropoietin (EPO) [1], leading to increased neurogenesis, generation of new tissue at the lesion site, and behavioural recovery. To translate similar therapies into the clinic, it is critical to evaluate, non-invasively and at multiple time points, this process of tissue repair and regeneration. In this study, we used a rat model of stroke to demonstrate the feasibility of using MR to distinguish between regenerating and pathological tissues when using endogenous stem cell therapies.
Experimental Cell Research
ABSTRACT Neural precursor cells (NPCs) respond to externally applied direct current electrical fi... more ABSTRACT Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non‐electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low‐Ca2+ culture conditions; an intracellular Ca2+ chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca2+ influx in DCEF‐induced NPC migration. Consistent with existing literature, we show Ca2+ is involved in F‐actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca2+ results in reduced velocity but has no effect on DCEF‐induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.
Biomaterials, 2016
Transplantation of pluripotent stem cells and their differentiated progeny has the potential to p... more Transplantation of pluripotent stem cells and their differentiated progeny has the potential to preserve or regenerate functional pathways and improve function after central nervous system injury. However, their utility has been hampered by poor survival and the potential to form tumors. Peptide-modified biomaterials influence cell adhesion, survival and differentiation in vitro, but their effectiveness in vivo remains uncertain. We synthesized a peptide-modified, minimally invasive, injectable hydrogel comprised of hyaluronan and methylcellulose to enhance the survival and differentiation of human induced pluripotent stem cell-derived oligodendrocyte progenitor cells. Cells were transplanted subacutely after a moderate clip compression rat spinal cord injury. The hydrogel, modified with the RGD peptide and platelet-derived growth factor (PDGF-A), promoted early survival and integration of grafted cells. However, prolific teratoma formation was evident when cells were transplanted in media at longer survival times, indicating that either this cell line or the way in which it was cultured is unsuitable for human use. Interestingly, teratoma formation was attenuated when cells were transplanted in the hydrogel, where most cells differentiated to a glial phenotype. Thus, this hydrogel promoted cell survival and integration, and attenuated teratoma formation by promoting cell differentiation.
Development, 1998
The adult mammalian forebrain contains a population of multipotential neural stem cells in the su... more The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average ...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the const... more The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-␣ (TGF␣) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23-to 25-months-old compared with 2-to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGF␣ does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGF␣ and the effects of senescence may regulate the proliferation of progenitor cells in the adult subependyma, but that the number of neural stem cells is maintained throughout life.
Stem Cell Research & Therapy, 2015
Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cell... more Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes)-a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny. Methods: NPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified. Results: We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation. Conclusion: We purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1992
The early development of the mammalian forebrain involves the massive proliferation of the ventri... more The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4....
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1997
The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the const... more The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-alpha (TGFalpha) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23- to 25-months-old compared with 2- to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGFalpha does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGFalpha and the effects of senescence may regulate the proliferation of progenitor cells in the adult subepe...
Development (Cambridge, England), 1998
The adult mammalian forebrain contains a population of multipotential neural stem cells in the su... more The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average ...
Disease Models & Mechanisms, 2014
Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neura... more 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 blocki...
![Research paper thumbnail of Satellite cell proliferation in the adult rat trigeminal ganglion results from the release of a mitogenic protein from explanted sensory neurons [published erratum appears in J Cell Biol 1994 Jun;125(6):1429]](https://attachments.academia-assets.com/75146999/thumbnails/1.jpg)
](The Journal of Cell Biology, 1994
Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of p... more Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of primarily satellite cells as opposed to Schwann cells. This proliferation begins at 15 h after explant culture and by 27 h there is a significant increase in glial proliferation as measured by scintillation counts of [3H]thymidine. Blocking protein synthesis between 0 and 3.5 h after explant culture (early) results in an enhanced proliferative response, while blocking protein synthesis between 3.5 and 7 h (late) causes a complete block of the proliferative response assessed at 27 h. Conditioned media experiments demonstrate that both the mitogenic and inhibitory signals are diffusible and heat labile. Finally, the addition of neurotrophic factors to rescue injured ganglionic neurons attenuates the proliferative glial response suggesting that injured neurons produce and release signals that induce glial proliferation.
Stroke, 2014
A ccelerating recovery in individuals who have experienced a stroke is crucial for limiting the e... more A ccelerating recovery in individuals who have experienced a stroke is crucial for limiting the effect of this condition. To enhance the rate of poststroke functional recovery, an optimal approach may be to combine rehabilitation with treatments that enhance neuroplasticity. Previous work has shown that serial application of epidermal growth factor (EGF) and erythropoietin enhances tissue regeneration and proliferation of neural precursor cells. 1 As a prelude to future mechanistic studies, the present study sought to determine whether an enhanced neuroplastic environment created by growth factor infusions and concurrent physical rehabilitation would augment behavioral recovery. We hypothesized that combining EGF and erythropoietin with rehabilitation after forelimb sensorimotor cortex stroke would result in accelerated functional improvements when compared with either rehabilitation or EGF and erythropoietin alone. Methods Subjects Sixty-seven male Sprague-Dawley rats were matched for poststroke grasping/retrieving performance on the staircase test and randomized into 4 groups: rehabilitation+EGF/erythropoietin (n=13), rehabilitation+artificial cerebrospinal fluid (aCSF; n=12), no rehabilitation+EGF/erythropoietin (n=12), and no rehabilitation+aCSF (n=13). All procedures were approved by the Memorial University Animal Care Committee and comply with regulations set by the Canadian Council of Animal Care (see detailed Methods in the online-only Data Supplement). Behavioral Testing Animals were trained on the staircase (pellet grasping/retrieving), beam (paw placement/balance), and cylinder (asymmetrical limb use for postural support) tasks 2 weeks before ischemia, and baseline abilities were assessed 3 days before stroke. Subsequent testing was performed 5 days after stroke and at poststroke weeks 4, 7, and 10 (Figure 1). Animals that did not meet preset training performance criteria (n=5), or were functionally unimpaired after stroke (n=12), were excluded. Behavioral data are presented as a percentage of prestroke performance. Surgical Procedures After baseline behavioral testing, rats were anesthetized with isoflurane and delivered 2 stereotaxic injections of endothelin-1 (2 μL/ Background and Purpose-Rehabilitation is the only treatment option for chronic stroke deficits, but unfortunately, it often provides incomplete recovery. In this study, a novel combination of growth factor administration and rehabilitation therapy was used to facilitate functional recovery in a rat model of cortical stroke. Methods-Ischemia was induced via injection of endothelin-1 into the sensorimotor cortex. This was followed by either a 2-week infusion of epidermal growth factor and erythropoietin or artificial cerebrospinal fluid into the ipsilateral lateral ventricle. Two weeks after ischemia, animals began an 8-week enriched rehabilitation program. Functional recovery was assessed after ischemia using the Montoya staircase-reaching task, beam-traversing, and cylinder test of forelimb asymmetry. Results-The combination of growth factor infusion and rehabilitation led to a significant acceleration in recovery in the staircase task. When compared with controls, animals receiving the combination treatment attained significant recovery of function at 4 weeks after stroke, whereas those receiving rehabilitation alone did not recover until 10 weeks. Significant recovery was also observed on the beam-traversing and cylinder tasks. Conclusions-Combining behavioral rehabilitation with growth factor infusion accelerates motor recovery. These data suggest a promising new avenue of combination therapies that may have the potential to reduce the rehabilitation time necessary to recover from sensorimotor deficits arising from stroke.
Neuroscience, 1999
Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal ... more Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a b-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically b-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the downregulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of b-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of b-galactosidase-labeled cells with [ 3 H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.
Journal of Magnetic Resonance Imaging, 2013
To evaluate the feasibility of using micron-sized superparamagnetic iron oxide particles (MPIOs) ... more To evaluate the feasibility of using micron-sized superparamagnetic iron oxide particles (MPIOs) as an effective labeling agent for monitoring bone marrow-derived mesenchymal stromal cell (BMSC) migration in the brain using magnetic resonance imaging (MRI) in a rat model of stroke and whether the accumulation of MPIO-labeled BMSCs can be differentiated from the accumulation of free MPIO particles or hemoglobin breakdown at a site of neuronal damage. In this study BMSCs were labeled with iron oxide and their pattern of migration following intravenous injection in a rat stroke model was monitored using a clinical MRI system followed by standard histopathology. The migration pattern was compared between intravenous injection of BMSCs alone, BMSCs labeled with MPIOs, and MPIO particles alone. The results demonstrated that while MRI was highly sensitive in the detection of iron oxide particle-containing cells in areas of neuronal ischemia, the true origin of cells containing iron oxide particles remains ambiguous. Therefore, detection of iron particles may not be a suitable strategy for the detection of BMSCs in the brain in a stroke model. This study suggests that the use of MPIOs as labeling agents are insufficient to conclusively determine the localization of iron within cells in regions of neuronal ischemia and hemorrhage.
Journal of Cerebral Blood Flow & Metabolism, 2006
Recent studies suggest that proliferation in the adult forebrain subventricular zone increases in... more Recent studies suggest that proliferation in the adult forebrain subventricular zone increases in response to a forebrain stroke and intraventricular infusions of growth factors enhance this response. The potential for growth factor infusions to regenerate the damaged motor cortex and promote recovery of motor function after stroke has not been examined. Here, we report that intraventricular infusions of epidermal growth factor and erythropoietin together, but not individually, promote substantial regeneration of the damaged cerebral cortex and reverse impairments in spontaneous and skilled motor tasks, in a rat model of stroke. Cortical regeneration and functional recovery occurred even when growth factor administration was delayed for up to 7 days after the stroke-induced lesion. Cell tracking demonstrated the contribution of neural precursors originating in the forebrain subventricular zone to the regenerated cortex. Strikingly, removal of the regenerated cortical tissue reversed...
Acta Pharmacologica Sinica, 2012
Stem cell-based therapies for the treatment of stroke have received considerable attention. Two b... more Stem cell-based therapies for the treatment of stroke have received considerable attention. Two broad approaches to stem cell-based therapies have been taken: the transplantation of exogenous stem cells, and the activation of endogenous neural stem and progenitor cells (together termed neural precursors). Studies examining the transplantation of exogenous cells have demonstrated that neural stem and progenitor cells lead to the most clinically promising results. Endogenous activation of neural precursors has also been explored based on the fact that resident precursor cells have the inherent capacity to proliferate, migrate and differentiate into mature neurons in the uninjured adult brain. Studies have revealed that these neural precursor cell behaviours can be activated following stroke, whereby neural precursors will expand in number, migrate to the infarct site and differentiate into neurons. However, this innate response is insufficient to lead to functional recovery, making it necessary to enhance the activation of endogenous precursors to promote tissue repair and functional recovery. Herein we will discuss the current state of the stem cell-based approaches with a focus on endogenous repair to treat the stroke injured brain.
Introduction: In animal models of stroke, endogenous neural precursor cells can be activated with... more Introduction: In animal models of stroke, endogenous neural precursor cells can be activated with growth factors such as epidermal growth factor (EGF) and erythropoietin (EPO) [1], leading to increased neurogenesis, generation of new tissue at the lesion site, and behavioural recovery. To translate similar therapies into the clinic, it is critical to evaluate, non-invasively and at multiple time points, this process of tissue repair and regeneration. In this study, we used a rat model of stroke to demonstrate the feasibility of using MR to distinguish between regenerating and pathological tissues when using endogenous stem cell therapies.
Experimental Cell Research
ABSTRACT Neural precursor cells (NPCs) respond to externally applied direct current electrical fi... more ABSTRACT Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non‐electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low‐Ca2+ culture conditions; an intracellular Ca2+ chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca2+ influx in DCEF‐induced NPC migration. Consistent with existing literature, we show Ca2+ is involved in F‐actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca2+ results in reduced velocity but has no effect on DCEF‐induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.
Biomaterials, 2016
Transplantation of pluripotent stem cells and their differentiated progeny has the potential to p... more Transplantation of pluripotent stem cells and their differentiated progeny has the potential to preserve or regenerate functional pathways and improve function after central nervous system injury. However, their utility has been hampered by poor survival and the potential to form tumors. Peptide-modified biomaterials influence cell adhesion, survival and differentiation in vitro, but their effectiveness in vivo remains uncertain. We synthesized a peptide-modified, minimally invasive, injectable hydrogel comprised of hyaluronan and methylcellulose to enhance the survival and differentiation of human induced pluripotent stem cell-derived oligodendrocyte progenitor cells. Cells were transplanted subacutely after a moderate clip compression rat spinal cord injury. The hydrogel, modified with the RGD peptide and platelet-derived growth factor (PDGF-A), promoted early survival and integration of grafted cells. However, prolific teratoma formation was evident when cells were transplanted in media at longer survival times, indicating that either this cell line or the way in which it was cultured is unsuitable for human use. Interestingly, teratoma formation was attenuated when cells were transplanted in the hydrogel, where most cells differentiated to a glial phenotype. Thus, this hydrogel promoted cell survival and integration, and attenuated teratoma formation by promoting cell differentiation.
Development, 1998
The adult mammalian forebrain contains a population of multipotential neural stem cells in the su... more The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average ...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the const... more The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-␣ (TGF␣) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23-to 25-months-old compared with 2-to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGF␣ does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGF␣ and the effects of senescence may regulate the proliferation of progenitor cells in the adult subependyma, but that the number of neural stem cells is maintained throughout life.
Stem Cell Research & Therapy, 2015
Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cell... more Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes)-a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny. Methods: NPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified. Results: We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation. Conclusion: We purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1992
The early development of the mammalian forebrain involves the massive proliferation of the ventri... more The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4....
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1997
The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the const... more The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-alpha (TGFalpha) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23- to 25-months-old compared with 2- to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGFalpha does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGFalpha and the effects of senescence may regulate the proliferation of progenitor cells in the adult subepe...
Development (Cambridge, England), 1998
The adult mammalian forebrain contains a population of multipotential neural stem cells in the su... more The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average ...
Disease Models & Mechanisms, 2014
Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neura... more 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 blocki...
The Journal of Cell Biology, 1994
Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of p... more Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of primarily satellite cells as opposed to Schwann cells. This proliferation begins at 15 h after explant culture and by 27 h there is a significant increase in glial proliferation as measured by scintillation counts of [3H]thymidine. Blocking protein synthesis between 0 and 3.5 h after explant culture (early) results in an enhanced proliferative response, while blocking protein synthesis between 3.5 and 7 h (late) causes a complete block of the proliferative response assessed at 27 h. Conditioned media experiments demonstrate that both the mitogenic and inhibitory signals are diffusible and heat labile. Finally, the addition of neurotrophic factors to rescue injured ganglionic neurons attenuates the proliferative glial response suggesting that injured neurons produce and release signals that induce glial proliferation.
Stroke, 2014
A ccelerating recovery in individuals who have experienced a stroke is crucial for limiting the e... more A ccelerating recovery in individuals who have experienced a stroke is crucial for limiting the effect of this condition. To enhance the rate of poststroke functional recovery, an optimal approach may be to combine rehabilitation with treatments that enhance neuroplasticity. Previous work has shown that serial application of epidermal growth factor (EGF) and erythropoietin enhances tissue regeneration and proliferation of neural precursor cells. 1 As a prelude to future mechanistic studies, the present study sought to determine whether an enhanced neuroplastic environment created by growth factor infusions and concurrent physical rehabilitation would augment behavioral recovery. We hypothesized that combining EGF and erythropoietin with rehabilitation after forelimb sensorimotor cortex stroke would result in accelerated functional improvements when compared with either rehabilitation or EGF and erythropoietin alone. Methods Subjects Sixty-seven male Sprague-Dawley rats were matched for poststroke grasping/retrieving performance on the staircase test and randomized into 4 groups: rehabilitation+EGF/erythropoietin (n=13), rehabilitation+artificial cerebrospinal fluid (aCSF; n=12), no rehabilitation+EGF/erythropoietin (n=12), and no rehabilitation+aCSF (n=13). All procedures were approved by the Memorial University Animal Care Committee and comply with regulations set by the Canadian Council of Animal Care (see detailed Methods in the online-only Data Supplement). Behavioral Testing Animals were trained on the staircase (pellet grasping/retrieving), beam (paw placement/balance), and cylinder (asymmetrical limb use for postural support) tasks 2 weeks before ischemia, and baseline abilities were assessed 3 days before stroke. Subsequent testing was performed 5 days after stroke and at poststroke weeks 4, 7, and 10 (Figure 1). Animals that did not meet preset training performance criteria (n=5), or were functionally unimpaired after stroke (n=12), were excluded. Behavioral data are presented as a percentage of prestroke performance. Surgical Procedures After baseline behavioral testing, rats were anesthetized with isoflurane and delivered 2 stereotaxic injections of endothelin-1 (2 μL/ Background and Purpose-Rehabilitation is the only treatment option for chronic stroke deficits, but unfortunately, it often provides incomplete recovery. In this study, a novel combination of growth factor administration and rehabilitation therapy was used to facilitate functional recovery in a rat model of cortical stroke. Methods-Ischemia was induced via injection of endothelin-1 into the sensorimotor cortex. This was followed by either a 2-week infusion of epidermal growth factor and erythropoietin or artificial cerebrospinal fluid into the ipsilateral lateral ventricle. Two weeks after ischemia, animals began an 8-week enriched rehabilitation program. Functional recovery was assessed after ischemia using the Montoya staircase-reaching task, beam-traversing, and cylinder test of forelimb asymmetry. Results-The combination of growth factor infusion and rehabilitation led to a significant acceleration in recovery in the staircase task. When compared with controls, animals receiving the combination treatment attained significant recovery of function at 4 weeks after stroke, whereas those receiving rehabilitation alone did not recover until 10 weeks. Significant recovery was also observed on the beam-traversing and cylinder tasks. Conclusions-Combining behavioral rehabilitation with growth factor infusion accelerates motor recovery. These data suggest a promising new avenue of combination therapies that may have the potential to reduce the rehabilitation time necessary to recover from sensorimotor deficits arising from stroke.
Neuroscience, 1999
Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal ... more Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a b-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically b-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the downregulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of b-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of b-galactosidase-labeled cells with [ 3 H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.