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Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

Abeysinghe HC, Bokhari L, Quigley A, Choolani M, Chan J, Dusting GJ, Crook JM, Kobayashi NR, Roulston CL - Stem Cell Res Ther (2015)

Bottom Line: Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission.Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells.In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar.

View Article: PubMed Central - PubMed

Affiliation: Neurotrauma Research Team, Department of Medicine, University of Melbourne, Level 4, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. himaa@student.unimelb.edu.au.

ABSTRACT

Introduction: Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain.

Methods: Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy.

Results: Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar.

Conclusion: Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

No MeSH data available.


Related in: MedlinePlus

Effect of treatment on endogenous neurogenesis and radial glial populations within the SVZ. Immunofluorescent images of proliferating cells (Ki67; red) and migrating immature neuroblasts (DCX; green) within the contralateral and ipsilateral SVZ of vehicle-treated (a, b), undifferentiated hNSC-treated (c, d), and predifferentiated cell-treated (e, f) animals. Nestin (green; arrowheads) and GFAP (red) immunopositive cells (coexpression giving a yellow appearance; arrows) within the contralateral and ipsilateral SVZ of vehicle-treated (g, h), undifferentiated treated (i, j), and predifferentiated treated (k, l) animals. All images were taken at the same anatomical location from animals with similar infarct volumes. Scale bar: (a–f) 500 μm, (g–l) 50 μm. CC corpus callosum, DCX doublecortin, GFAP glial fibrillary acidic protein, LV lateral ventricle, Prediff predifferentiated treated, Stm Striatum, Undiff undifferentiated hNSC treated
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Fig8: Effect of treatment on endogenous neurogenesis and radial glial populations within the SVZ. Immunofluorescent images of proliferating cells (Ki67; red) and migrating immature neuroblasts (DCX; green) within the contralateral and ipsilateral SVZ of vehicle-treated (a, b), undifferentiated hNSC-treated (c, d), and predifferentiated cell-treated (e, f) animals. Nestin (green; arrowheads) and GFAP (red) immunopositive cells (coexpression giving a yellow appearance; arrows) within the contralateral and ipsilateral SVZ of vehicle-treated (g, h), undifferentiated treated (i, j), and predifferentiated treated (k, l) animals. All images were taken at the same anatomical location from animals with similar infarct volumes. Scale bar: (a–f) 500 μm, (g–l) 50 μm. CC corpus callosum, DCX doublecortin, GFAP glial fibrillary acidic protein, LV lateral ventricle, Prediff predifferentiated treated, Stm Striatum, Undiff undifferentiated hNSC treated

Mentions: To investigate the effects of SVZ-hNSC transplant on endogenous recovery mechanisms we examined changes within the neurogenic niche in response to stem cell transplant. Confocal analysis revealed an apparent increase in the number of Ki67-positive proliferating cells within the ipsilateral SVZ of rats that received predifferentiated SVZ-hNSCs in comparison with rats that received undifferentiated SVZ-hNSCs or vehicle controls (Fig. 8a–f). In particular, immunofluorescent labeling of cells within the SVZ revealed an apparent increase in the number of newly generated DCX-positive neurons in rats with predifferentiated SVZ-hNSC transplants in comparison with the contralateral SVZ, and rats with undifferentiated hNSC transplants or vehicle controls (Fig. 8b, d, f). Further analysis of immunolabeled cells within the SVZ revealed an apparent increase in the number of GFAP-positive radial glial cells with long processes directed towards the infarct that did not colocalize with Nestin in animals that received predifferentiated SVZ-hNSC transplants compared with the contralateral SVZ, and compared with the other treatment groups (Fig. 8g–l). Furthermore, cells identified within the neurogenic niche of the SVZ were not immunopositive for HuNu, a marker of hNSCs.Fig. 8


Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

Abeysinghe HC, Bokhari L, Quigley A, Choolani M, Chan J, Dusting GJ, Crook JM, Kobayashi NR, Roulston CL - Stem Cell Res Ther (2015)

Effect of treatment on endogenous neurogenesis and radial glial populations within the SVZ. Immunofluorescent images of proliferating cells (Ki67; red) and migrating immature neuroblasts (DCX; green) within the contralateral and ipsilateral SVZ of vehicle-treated (a, b), undifferentiated hNSC-treated (c, d), and predifferentiated cell-treated (e, f) animals. Nestin (green; arrowheads) and GFAP (red) immunopositive cells (coexpression giving a yellow appearance; arrows) within the contralateral and ipsilateral SVZ of vehicle-treated (g, h), undifferentiated treated (i, j), and predifferentiated treated (k, l) animals. All images were taken at the same anatomical location from animals with similar infarct volumes. Scale bar: (a–f) 500 μm, (g–l) 50 μm. CC corpus callosum, DCX doublecortin, GFAP glial fibrillary acidic protein, LV lateral ventricle, Prediff predifferentiated treated, Stm Striatum, Undiff undifferentiated hNSC treated
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig8: Effect of treatment on endogenous neurogenesis and radial glial populations within the SVZ. Immunofluorescent images of proliferating cells (Ki67; red) and migrating immature neuroblasts (DCX; green) within the contralateral and ipsilateral SVZ of vehicle-treated (a, b), undifferentiated hNSC-treated (c, d), and predifferentiated cell-treated (e, f) animals. Nestin (green; arrowheads) and GFAP (red) immunopositive cells (coexpression giving a yellow appearance; arrows) within the contralateral and ipsilateral SVZ of vehicle-treated (g, h), undifferentiated treated (i, j), and predifferentiated treated (k, l) animals. All images were taken at the same anatomical location from animals with similar infarct volumes. Scale bar: (a–f) 500 μm, (g–l) 50 μm. CC corpus callosum, DCX doublecortin, GFAP glial fibrillary acidic protein, LV lateral ventricle, Prediff predifferentiated treated, Stm Striatum, Undiff undifferentiated hNSC treated
Mentions: To investigate the effects of SVZ-hNSC transplant on endogenous recovery mechanisms we examined changes within the neurogenic niche in response to stem cell transplant. Confocal analysis revealed an apparent increase in the number of Ki67-positive proliferating cells within the ipsilateral SVZ of rats that received predifferentiated SVZ-hNSCs in comparison with rats that received undifferentiated SVZ-hNSCs or vehicle controls (Fig. 8a–f). In particular, immunofluorescent labeling of cells within the SVZ revealed an apparent increase in the number of newly generated DCX-positive neurons in rats with predifferentiated SVZ-hNSC transplants in comparison with the contralateral SVZ, and rats with undifferentiated hNSC transplants or vehicle controls (Fig. 8b, d, f). Further analysis of immunolabeled cells within the SVZ revealed an apparent increase in the number of GFAP-positive radial glial cells with long processes directed towards the infarct that did not colocalize with Nestin in animals that received predifferentiated SVZ-hNSC transplants compared with the contralateral SVZ, and compared with the other treatment groups (Fig. 8g–l). Furthermore, cells identified within the neurogenic niche of the SVZ were not immunopositive for HuNu, a marker of hNSCs.Fig. 8

Bottom Line: Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission.Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells.In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar.

View Article: PubMed Central - PubMed

Affiliation: Neurotrauma Research Team, Department of Medicine, University of Melbourne, Level 4, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. himaa@student.unimelb.edu.au.

ABSTRACT

Introduction: Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain.

Methods: Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy.

Results: Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar.

Conclusion: Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

No MeSH data available.


Related in: MedlinePlus