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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

Phenotypic profile of transplanted undifferentiated hNSCs versus predifferentiated cells. Coronal sections immunostained with NeuN (green) highlight undamaged brain regions with lack of staining within stroke affected areas (white dotted line) at cortical graft sites +1.4 mm and –2.16 mm relative to the bregma (a, c) and striatal graft sites +1.4 mm and +0.2 mm relative to the bregma (e, g); white boxes depict graft location. Representative images of graft sites stained with HuNu (red) and NeuN (green) (b, d, f, h) from regions highlighted by white boxes (a, c, e, g). Numbers of HuNu-positive cells coexpressing Tuj1, GABA, Nestin, GFAP, and Ki67 from undifferentiated hNSC-treated (n = 7) and predifferentiated cell-treated (n = 7) animals from cortical graft sites; +1.4 mm (i) and –2.16 mm (j) relative to the bregma; and striatal graft sites +1.4 mm (k) and +0.2 mm (l) relative to the bregma. Numbers of cells are presented as a percentage of the total number of HuNu-positive cells counted. Data are mean ± SEM. ***P <0.001, ****P <0.0001 compared with undifferentiated hNSC counts (two-way ANOVA with Bonferroni post test). Scale bar: (a, c, e, g) 2000 μm, (b, d, f, h) 200 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, NeuN neuron specific nuclear antigen, Tuj1 β-III tubulin
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Fig5: Phenotypic profile of transplanted undifferentiated hNSCs versus predifferentiated cells. Coronal sections immunostained with NeuN (green) highlight undamaged brain regions with lack of staining within stroke affected areas (white dotted line) at cortical graft sites +1.4 mm and –2.16 mm relative to the bregma (a, c) and striatal graft sites +1.4 mm and +0.2 mm relative to the bregma (e, g); white boxes depict graft location. Representative images of graft sites stained with HuNu (red) and NeuN (green) (b, d, f, h) from regions highlighted by white boxes (a, c, e, g). Numbers of HuNu-positive cells coexpressing Tuj1, GABA, Nestin, GFAP, and Ki67 from undifferentiated hNSC-treated (n = 7) and predifferentiated cell-treated (n = 7) animals from cortical graft sites; +1.4 mm (i) and –2.16 mm (j) relative to the bregma; and striatal graft sites +1.4 mm (k) and +0.2 mm (l) relative to the bregma. Numbers of cells are presented as a percentage of the total number of HuNu-positive cells counted. Data are mean ± SEM. ***P <0.001, ****P <0.0001 compared with undifferentiated hNSC counts (two-way ANOVA with Bonferroni post test). Scale bar: (a, c, e, g) 2000 μm, (b, d, f, h) 200 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, NeuN neuron specific nuclear antigen, Tuj1 β-III tubulin

Mentions: Immunofluorescent NeuN staining was used to confirm positioning of graft sites within the infarcted region for stereological analysis across all groups receiving SVZ-hNSC transplants (Fig. 5a–h). Cell counts within each site revealed that there were significantly less cells that expressed Tuj1 (6.6 ± 1.3 %; P <0.0001) and GABA (5.4 ± 1.4 %; P <0.0001) for undifferentiated SVZ-hNSC grafts compared with predifferentiated cell grafts (Tuj1: 89.6 ± 1.9 %; GABA: 94.3 ± 1.6 %) across all transplant sites (Fig. 5i–l, ANOVA). Conversely, there was a significantly greater proportion of undifferentiated SVZ-hNSCs that expressed markers for GFAP (93.0 ± 1.9 %; P <0.0001), Nestin (88.1 ± 2.8 %; P <0.0001), and Ki67 (11.9 ± 1.6 %; P <0.05) compared with transplanted predifferentiated cells (GFAP: 4.7 ± 1.3 %; Nestin: 11.6 ± 3.3 %; Ki67: 4.6 ± 1.1 %) (Fig. 5i–l).Fig. 5


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)

Phenotypic profile of transplanted undifferentiated hNSCs versus predifferentiated cells. Coronal sections immunostained with NeuN (green) highlight undamaged brain regions with lack of staining within stroke affected areas (white dotted line) at cortical graft sites +1.4 mm and –2.16 mm relative to the bregma (a, c) and striatal graft sites +1.4 mm and +0.2 mm relative to the bregma (e, g); white boxes depict graft location. Representative images of graft sites stained with HuNu (red) and NeuN (green) (b, d, f, h) from regions highlighted by white boxes (a, c, e, g). Numbers of HuNu-positive cells coexpressing Tuj1, GABA, Nestin, GFAP, and Ki67 from undifferentiated hNSC-treated (n = 7) and predifferentiated cell-treated (n = 7) animals from cortical graft sites; +1.4 mm (i) and –2.16 mm (j) relative to the bregma; and striatal graft sites +1.4 mm (k) and +0.2 mm (l) relative to the bregma. Numbers of cells are presented as a percentage of the total number of HuNu-positive cells counted. Data are mean ± SEM. ***P <0.001, ****P <0.0001 compared with undifferentiated hNSC counts (two-way ANOVA with Bonferroni post test). Scale bar: (a, c, e, g) 2000 μm, (b, d, f, h) 200 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, NeuN neuron specific nuclear antigen, Tuj1 β-III tubulin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4588906&req=5

Fig5: Phenotypic profile of transplanted undifferentiated hNSCs versus predifferentiated cells. Coronal sections immunostained with NeuN (green) highlight undamaged brain regions with lack of staining within stroke affected areas (white dotted line) at cortical graft sites +1.4 mm and –2.16 mm relative to the bregma (a, c) and striatal graft sites +1.4 mm and +0.2 mm relative to the bregma (e, g); white boxes depict graft location. Representative images of graft sites stained with HuNu (red) and NeuN (green) (b, d, f, h) from regions highlighted by white boxes (a, c, e, g). Numbers of HuNu-positive cells coexpressing Tuj1, GABA, Nestin, GFAP, and Ki67 from undifferentiated hNSC-treated (n = 7) and predifferentiated cell-treated (n = 7) animals from cortical graft sites; +1.4 mm (i) and –2.16 mm (j) relative to the bregma; and striatal graft sites +1.4 mm (k) and +0.2 mm (l) relative to the bregma. Numbers of cells are presented as a percentage of the total number of HuNu-positive cells counted. Data are mean ± SEM. ***P <0.001, ****P <0.0001 compared with undifferentiated hNSC counts (two-way ANOVA with Bonferroni post test). Scale bar: (a, c, e, g) 2000 μm, (b, d, f, h) 200 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, NeuN neuron specific nuclear antigen, Tuj1 β-III tubulin
Mentions: Immunofluorescent NeuN staining was used to confirm positioning of graft sites within the infarcted region for stereological analysis across all groups receiving SVZ-hNSC transplants (Fig. 5a–h). Cell counts within each site revealed that there were significantly less cells that expressed Tuj1 (6.6 ± 1.3 %; P <0.0001) and GABA (5.4 ± 1.4 %; P <0.0001) for undifferentiated SVZ-hNSC grafts compared with predifferentiated cell grafts (Tuj1: 89.6 ± 1.9 %; GABA: 94.3 ± 1.6 %) across all transplant sites (Fig. 5i–l, ANOVA). Conversely, there was a significantly greater proportion of undifferentiated SVZ-hNSCs that expressed markers for GFAP (93.0 ± 1.9 %; P <0.0001), Nestin (88.1 ± 2.8 %; P <0.0001), and Ki67 (11.9 ± 1.6 %; P <0.05) compared with transplanted predifferentiated cells (GFAP: 4.7 ± 1.3 %; Nestin: 11.6 ± 3.3 %; Ki67: 4.6 ± 1.1 %) (Fig. 5i–l).Fig. 5

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