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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

Transplanted cells survive within the stroke affected brain. Total number (a) and percentage (b) of HuNu-positive cells remaining within cortical and striatal grafts from undifferentiated (n = 7) and predifferentiated (n = 7) treatment groups. Data presented as mean ± SEM. ****P <0.0001 relative to undifferentiated treated animals in the same region; ####P <0.0001 relative to undifferentiated grafts within the cortex; ϕP <0.05 relative to predifferentiated grafts within the striatum (two-way ANOVA followed by Bonferroni post test). Transplanted cells immunopositive for HuNu (red) (c, f) did not express apoptotic markers including Casp3 (green) (d), merged image (e), or TUNEL (g), with the level of TUNEL staining similar to the contralateral mirror image (h). Transplanted HuNu-positive (red) predifferentiated cells were associated with vWF-stained blood vessels (green) within infarcted brain regions (i). Many HuNu-positive undifferentiated hNSCs (red) were found within border regions consisting of GFAP-positive astrocytes (green) (j). Magnified immunofluorescent image (k) corresponds to box highlighted in (j) illustrating incorporation of undifferentiated hNSCs into the glial scar bordering the infarct. Scale bar: (c–e) 20 μm, (f–h) 100 μm, (i, k) 200 μm, (j) 100 μm. C contralateral hemisphere, Casp3 cleaved caspase-3, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, I ipsilateral hemisphere, TUNEL terminal transferase-mediated dUTP nick end-labeling, vWF von Willebrand factor
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Fig3: Transplanted cells survive within the stroke affected brain. Total number (a) and percentage (b) of HuNu-positive cells remaining within cortical and striatal grafts from undifferentiated (n = 7) and predifferentiated (n = 7) treatment groups. Data presented as mean ± SEM. ****P <0.0001 relative to undifferentiated treated animals in the same region; ####P <0.0001 relative to undifferentiated grafts within the cortex; ϕP <0.05 relative to predifferentiated grafts within the striatum (two-way ANOVA followed by Bonferroni post test). Transplanted cells immunopositive for HuNu (red) (c, f) did not express apoptotic markers including Casp3 (green) (d), merged image (e), or TUNEL (g), with the level of TUNEL staining similar to the contralateral mirror image (h). Transplanted HuNu-positive (red) predifferentiated cells were associated with vWF-stained blood vessels (green) within infarcted brain regions (i). Many HuNu-positive undifferentiated hNSCs (red) were found within border regions consisting of GFAP-positive astrocytes (green) (j). Magnified immunofluorescent image (k) corresponds to box highlighted in (j) illustrating incorporation of undifferentiated hNSCs into the glial scar bordering the infarct. Scale bar: (c–e) 20 μm, (f–h) 100 μm, (i, k) 200 μm, (j) 100 μm. C contralateral hemisphere, Casp3 cleaved caspase-3, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, I ipsilateral hemisphere, TUNEL terminal transferase-mediated dUTP nick end-labeling, vWF von Willebrand factor

Mentions: HuNu immunostaining revealed large numbers of SVZ-hNSCs within each graft site situated both within and outside the damaged regions 28 days post transplant. Although rats receiving undifferentiated hNSCs displayed evidence of cells within each transplant site, many HuNu-positive NSCs were also detected beyond the transplant regions and were also visible within the infarct border zone. Rats that received predifferentiated SVZ-hNSCs showed dense clustering of cells within each site. Stereological cell counting revealed significantly fewer undifferentiated SVZ-hNSCs within the cortex and striatum (64,712 ± 16,866 cells and 260,278 ± 14,112 cells respectively) compared with predifferentiated HuNu-positive cells in cortical and striatal graft regions (444,852 ± 22,181 and 508,098 ± 10,031 cells respectively) (P <0.0001; Fig. 3a). A greater number of predifferentiated (P <0.05) and undifferentiated (P <0.0001) SVZ-hNSCs remained within striatal grafts compared with cortical graft sites (Fig. 3a). However the overall percentage of transplanted predifferentiated cells remaining within cortical and striatal graft sites (27.8 ± 1.3 % and 31.8 ± 0.5 % respectively) was significantly higher than that of undifferentiated SVZ-hNSCs within the cortex and striatum (4.0 ± 1.0 % and 16.3 ± 0.9 % respectively) (P <0.0001; Fig. 3b).Fig. 3


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)

Transplanted cells survive within the stroke affected brain. Total number (a) and percentage (b) of HuNu-positive cells remaining within cortical and striatal grafts from undifferentiated (n = 7) and predifferentiated (n = 7) treatment groups. Data presented as mean ± SEM. ****P <0.0001 relative to undifferentiated treated animals in the same region; ####P <0.0001 relative to undifferentiated grafts within the cortex; ϕP <0.05 relative to predifferentiated grafts within the striatum (two-way ANOVA followed by Bonferroni post test). Transplanted cells immunopositive for HuNu (red) (c, f) did not express apoptotic markers including Casp3 (green) (d), merged image (e), or TUNEL (g), with the level of TUNEL staining similar to the contralateral mirror image (h). Transplanted HuNu-positive (red) predifferentiated cells were associated with vWF-stained blood vessels (green) within infarcted brain regions (i). Many HuNu-positive undifferentiated hNSCs (red) were found within border regions consisting of GFAP-positive astrocytes (green) (j). Magnified immunofluorescent image (k) corresponds to box highlighted in (j) illustrating incorporation of undifferentiated hNSCs into the glial scar bordering the infarct. Scale bar: (c–e) 20 μm, (f–h) 100 μm, (i, k) 200 μm, (j) 100 μm. C contralateral hemisphere, Casp3 cleaved caspase-3, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, I ipsilateral hemisphere, TUNEL terminal transferase-mediated dUTP nick end-labeling, vWF von Willebrand factor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588906&req=5

Fig3: Transplanted cells survive within the stroke affected brain. Total number (a) and percentage (b) of HuNu-positive cells remaining within cortical and striatal grafts from undifferentiated (n = 7) and predifferentiated (n = 7) treatment groups. Data presented as mean ± SEM. ****P <0.0001 relative to undifferentiated treated animals in the same region; ####P <0.0001 relative to undifferentiated grafts within the cortex; ϕP <0.05 relative to predifferentiated grafts within the striatum (two-way ANOVA followed by Bonferroni post test). Transplanted cells immunopositive for HuNu (red) (c, f) did not express apoptotic markers including Casp3 (green) (d), merged image (e), or TUNEL (g), with the level of TUNEL staining similar to the contralateral mirror image (h). Transplanted HuNu-positive (red) predifferentiated cells were associated with vWF-stained blood vessels (green) within infarcted brain regions (i). Many HuNu-positive undifferentiated hNSCs (red) were found within border regions consisting of GFAP-positive astrocytes (green) (j). Magnified immunofluorescent image (k) corresponds to box highlighted in (j) illustrating incorporation of undifferentiated hNSCs into the glial scar bordering the infarct. Scale bar: (c–e) 20 μm, (f–h) 100 μm, (i, k) 200 μm, (j) 100 μm. C contralateral hemisphere, Casp3 cleaved caspase-3, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, I ipsilateral hemisphere, TUNEL terminal transferase-mediated dUTP nick end-labeling, vWF von Willebrand factor
Mentions: HuNu immunostaining revealed large numbers of SVZ-hNSCs within each graft site situated both within and outside the damaged regions 28 days post transplant. Although rats receiving undifferentiated hNSCs displayed evidence of cells within each transplant site, many HuNu-positive NSCs were also detected beyond the transplant regions and were also visible within the infarct border zone. Rats that received predifferentiated SVZ-hNSCs showed dense clustering of cells within each site. Stereological cell counting revealed significantly fewer undifferentiated SVZ-hNSCs within the cortex and striatum (64,712 ± 16,866 cells and 260,278 ± 14,112 cells respectively) compared with predifferentiated HuNu-positive cells in cortical and striatal graft regions (444,852 ± 22,181 and 508,098 ± 10,031 cells respectively) (P <0.0001; Fig. 3a). A greater number of predifferentiated (P <0.05) and undifferentiated (P <0.0001) SVZ-hNSCs remained within striatal grafts compared with cortical graft sites (Fig. 3a). However the overall percentage of transplanted predifferentiated cells remaining within cortical and striatal graft sites (27.8 ± 1.3 % and 31.8 ± 0.5 % respectively) was significantly higher than that of undifferentiated SVZ-hNSCs within the cortex and striatum (4.0 ± 1.0 % and 16.3 ± 0.9 % respectively) (P <0.0001; Fig. 3b).Fig. 3

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