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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

Functional outcome following transplantation. Effects of transplantation on contralateral limb use when rearing (a) in the cylinder test after ET-1-induced stroke. Data are mean ± standard error of the mean (SEM) expressed as a percentage of contralateral (impaired) forelimb use. Each rat acted as its own control; results following stroke were compared with 0-hour prestroke scores. ϕP <0.05, ϕϕP <0.01, ϕϕϕϕP <0.0001 relative to 0-hour poststroke baseline scores for vehicle-treated rats (n = 7); **P <0.01, ***P <0.001, ****P <0.0001 compared with 0-hour poststroke baseline scores for undifferentiated treated rats (n = 7); #P <0.05, ##P <0.01 relative to 0-hour poststroke baseline scores for predifferentiated treated rats (n = 7); δP <0.05, δδP <0.01 vehicle-treated rats compared with predifferentiated treated rats; φφP <0.01 vehicle-treated rats compared with undifferentiated treated rats; †P <0.05 predifferentiated treated rats compared with undifferentiated treated rats (two-way ANOVA followed by Bonferroni post test). Combined neurological deficit scores (b). Data presented as box plots include hinges extending from the 25th to 75th percentiles, the median line within the box and whiskers extending to the minimum and maximum values of the dataset (n = 7/group). *P <0.05, ***P <0.001, ****P <0.0001 relative to 0-hour poststroke baseline scores (n = 7/group); #P <0.05, ##P <0.01, ###P <0.001, ####P <0.0001 relative to pretransplant scores (Kruskal–Wallis ANOVA followed by Dunn’s test). Effect of transplanting vehicle, undifferentiated hNSCs, or predifferentiated cells on infarct area (c), (d) and total infarct volume (e) within the cortex and striatum. Data presented as mean ± SEM of infarct area measured at eight predetermined coronal planes through the brain (two-way ANOVA followed by Bonferroni post test)
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Fig2: Functional outcome following transplantation. Effects of transplantation on contralateral limb use when rearing (a) in the cylinder test after ET-1-induced stroke. Data are mean ± standard error of the mean (SEM) expressed as a percentage of contralateral (impaired) forelimb use. Each rat acted as its own control; results following stroke were compared with 0-hour prestroke scores. ϕP <0.05, ϕϕP <0.01, ϕϕϕϕP <0.0001 relative to 0-hour poststroke baseline scores for vehicle-treated rats (n = 7); **P <0.01, ***P <0.001, ****P <0.0001 compared with 0-hour poststroke baseline scores for undifferentiated treated rats (n = 7); #P <0.05, ##P <0.01 relative to 0-hour poststroke baseline scores for predifferentiated treated rats (n = 7); δP <0.05, δδP <0.01 vehicle-treated rats compared with predifferentiated treated rats; φφP <0.01 vehicle-treated rats compared with undifferentiated treated rats; †P <0.05 predifferentiated treated rats compared with undifferentiated treated rats (two-way ANOVA followed by Bonferroni post test). Combined neurological deficit scores (b). Data presented as box plots include hinges extending from the 25th to 75th percentiles, the median line within the box and whiskers extending to the minimum and maximum values of the dataset (n = 7/group). *P <0.05, ***P <0.001, ****P <0.0001 relative to 0-hour poststroke baseline scores (n = 7/group); #P <0.05, ##P <0.01, ###P <0.001, ####P <0.0001 relative to pretransplant scores (Kruskal–Wallis ANOVA followed by Dunn’s test). Effect of transplanting vehicle, undifferentiated hNSCs, or predifferentiated cells on infarct area (c), (d) and total infarct volume (e) within the cortex and striatum. Data presented as mean ± SEM of infarct area measured at eight predetermined coronal planes through the brain (two-way ANOVA followed by Bonferroni post test)

Mentions: No significant bias in forelimb use upon rearing was detected using the cylinder test prior to stroke induction in any treatment group. After stroke, all rats exhibited asymmetrical limb use indicative of stroke damage with preferential ipsilateral (unimpaired) forelimb use during rearing (Fig. 2a). While transplantation of either undifferentiated SVZ-hNSCs and predifferentiated SVZ-hNSCs appeared to decrease forelimb asymmetry after treatment, this effect was only statistically significant in rats receiving predifferentiated hNSCs at 7 and 28 days post transplant in comparison with vehicle control rats receiving media alone (Fig. 2a) (P <0.05, two-way ANOVA).Fig. 2


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)

Functional outcome following transplantation. Effects of transplantation on contralateral limb use when rearing (a) in the cylinder test after ET-1-induced stroke. Data are mean ± standard error of the mean (SEM) expressed as a percentage of contralateral (impaired) forelimb use. Each rat acted as its own control; results following stroke were compared with 0-hour prestroke scores. ϕP <0.05, ϕϕP <0.01, ϕϕϕϕP <0.0001 relative to 0-hour poststroke baseline scores for vehicle-treated rats (n = 7); **P <0.01, ***P <0.001, ****P <0.0001 compared with 0-hour poststroke baseline scores for undifferentiated treated rats (n = 7); #P <0.05, ##P <0.01 relative to 0-hour poststroke baseline scores for predifferentiated treated rats (n = 7); δP <0.05, δδP <0.01 vehicle-treated rats compared with predifferentiated treated rats; φφP <0.01 vehicle-treated rats compared with undifferentiated treated rats; †P <0.05 predifferentiated treated rats compared with undifferentiated treated rats (two-way ANOVA followed by Bonferroni post test). Combined neurological deficit scores (b). Data presented as box plots include hinges extending from the 25th to 75th percentiles, the median line within the box and whiskers extending to the minimum and maximum values of the dataset (n = 7/group). *P <0.05, ***P <0.001, ****P <0.0001 relative to 0-hour poststroke baseline scores (n = 7/group); #P <0.05, ##P <0.01, ###P <0.001, ####P <0.0001 relative to pretransplant scores (Kruskal–Wallis ANOVA followed by Dunn’s test). Effect of transplanting vehicle, undifferentiated hNSCs, or predifferentiated cells on infarct area (c), (d) and total infarct volume (e) within the cortex and striatum. Data presented as mean ± SEM of infarct area measured at eight predetermined coronal planes through the brain (two-way ANOVA followed by Bonferroni post test)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Functional outcome following transplantation. Effects of transplantation on contralateral limb use when rearing (a) in the cylinder test after ET-1-induced stroke. Data are mean ± standard error of the mean (SEM) expressed as a percentage of contralateral (impaired) forelimb use. Each rat acted as its own control; results following stroke were compared with 0-hour prestroke scores. ϕP <0.05, ϕϕP <0.01, ϕϕϕϕP <0.0001 relative to 0-hour poststroke baseline scores for vehicle-treated rats (n = 7); **P <0.01, ***P <0.001, ****P <0.0001 compared with 0-hour poststroke baseline scores for undifferentiated treated rats (n = 7); #P <0.05, ##P <0.01 relative to 0-hour poststroke baseline scores for predifferentiated treated rats (n = 7); δP <0.05, δδP <0.01 vehicle-treated rats compared with predifferentiated treated rats; φφP <0.01 vehicle-treated rats compared with undifferentiated treated rats; †P <0.05 predifferentiated treated rats compared with undifferentiated treated rats (two-way ANOVA followed by Bonferroni post test). Combined neurological deficit scores (b). Data presented as box plots include hinges extending from the 25th to 75th percentiles, the median line within the box and whiskers extending to the minimum and maximum values of the dataset (n = 7/group). *P <0.05, ***P <0.001, ****P <0.0001 relative to 0-hour poststroke baseline scores (n = 7/group); #P <0.05, ##P <0.01, ###P <0.001, ####P <0.0001 relative to pretransplant scores (Kruskal–Wallis ANOVA followed by Dunn’s test). Effect of transplanting vehicle, undifferentiated hNSCs, or predifferentiated cells on infarct area (c), (d) and total infarct volume (e) within the cortex and striatum. Data presented as mean ± SEM of infarct area measured at eight predetermined coronal planes through the brain (two-way ANOVA followed by Bonferroni post test)
Mentions: No significant bias in forelimb use upon rearing was detected using the cylinder test prior to stroke induction in any treatment group. After stroke, all rats exhibited asymmetrical limb use indicative of stroke damage with preferential ipsilateral (unimpaired) forelimb use during rearing (Fig. 2a). While transplantation of either undifferentiated SVZ-hNSCs and predifferentiated SVZ-hNSCs appeared to decrease forelimb asymmetry after treatment, this effect was only statistically significant in rats receiving predifferentiated hNSCs at 7 and 28 days post transplant in comparison with vehicle control rats receiving media alone (Fig. 2a) (P <0.05, two-way ANOVA).Fig. 2

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