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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

Further maturation of predifferentiated cells 28 days post transplant. Confocal photomicrographs of predifferentiated cells 28 days post transplant within the stroke-damaged brain expressed HuNu (red) double labeled with either GAD65/67 (green) (a), calbindin-D28k (CB; green) (b), or calretinin (CR, green) (c). Predifferentiated cells expressing HuNu (blue) triple-labeled with parvalbumin (PV, green) and GABA (red) (d), or triple-labeled with Tuj1 (red) and presynaptic vesicle protein synaptophysin (SYN, green) within the cortical core (e) and border (f) regions. Some predifferentiated cells expressing HuNu (red) grafted to border regions appeared to extend long neurites expressing Tuj1 (green) (g). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. Scale bar: (a, b) 10 μm, orthogonal image 5 μm; (c, d) 40 μm, orthogonal image 5 μm; (e, f) 10 μm, orthogonal image 5 μm; (g) 20 μm, orthogonal image 5 μm. GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
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Fig6: Further maturation of predifferentiated cells 28 days post transplant. Confocal photomicrographs of predifferentiated cells 28 days post transplant within the stroke-damaged brain expressed HuNu (red) double labeled with either GAD65/67 (green) (a), calbindin-D28k (CB; green) (b), or calretinin (CR, green) (c). Predifferentiated cells expressing HuNu (blue) triple-labeled with parvalbumin (PV, green) and GABA (red) (d), or triple-labeled with Tuj1 (red) and presynaptic vesicle protein synaptophysin (SYN, green) within the cortical core (e) and border (f) regions. Some predifferentiated cells expressing HuNu (red) grafted to border regions appeared to extend long neurites expressing Tuj1 (green) (g). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. Scale bar: (a, b) 10 μm, orthogonal image 5 μm; (c, d) 40 μm, orthogonal image 5 μm; (e, f) 10 μm, orthogonal image 5 μm; (g) 20 μm, orthogonal image 5 μm. GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin

Mentions: Further confocal analysis revealed that predifferentiated SVZ-hNSC grafts expressed GAD65/67 (Fig. 6a), as well as the intracellular CBPs CB (Fig. 6b), CR (Fig. 6c), and PV (Fig. 6d). In addition, predifferentiated SVZ-hNSCs were observed to express SYN that was mostly localized to the cell body of cells transplanted into the core infarct region (Fig. 6e), but was also additionally observed along the cytoskeleton of cells that were transplanted into sites outside of the infarct within the peri-infarct territory (Fig. 6f). Predifferentiated SVZ-hNSC grafts located within the peri-infarct territory appeared to extend long neurites revealed by HuNu and Tuj1 immunostaining (Fig. 6g). Grafts located within the core infarct were surrounded by the glial scar consisting of densely packed GFAP-positive astrocytes, with limited dispersion of SVZ-hNSCs in closest proximity to the scar (Fig. 7a–c). Parallel negative control experiments omitting primary antibodies depict a low level of autofluorescence within the core infarct (Fig. d–f).Fig. 6


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)

Further maturation of predifferentiated cells 28 days post transplant. Confocal photomicrographs of predifferentiated cells 28 days post transplant within the stroke-damaged brain expressed HuNu (red) double labeled with either GAD65/67 (green) (a), calbindin-D28k (CB; green) (b), or calretinin (CR, green) (c). Predifferentiated cells expressing HuNu (blue) triple-labeled with parvalbumin (PV, green) and GABA (red) (d), or triple-labeled with Tuj1 (red) and presynaptic vesicle protein synaptophysin (SYN, green) within the cortical core (e) and border (f) regions. Some predifferentiated cells expressing HuNu (red) grafted to border regions appeared to extend long neurites expressing Tuj1 (green) (g). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. Scale bar: (a, b) 10 μm, orthogonal image 5 μm; (c, d) 40 μm, orthogonal image 5 μm; (e, f) 10 μm, orthogonal image 5 μm; (g) 20 μm, orthogonal image 5 μm. GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
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Fig6: Further maturation of predifferentiated cells 28 days post transplant. Confocal photomicrographs of predifferentiated cells 28 days post transplant within the stroke-damaged brain expressed HuNu (red) double labeled with either GAD65/67 (green) (a), calbindin-D28k (CB; green) (b), or calretinin (CR, green) (c). Predifferentiated cells expressing HuNu (blue) triple-labeled with parvalbumin (PV, green) and GABA (red) (d), or triple-labeled with Tuj1 (red) and presynaptic vesicle protein synaptophysin (SYN, green) within the cortical core (e) and border (f) regions. Some predifferentiated cells expressing HuNu (red) grafted to border regions appeared to extend long neurites expressing Tuj1 (green) (g). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. Scale bar: (a, b) 10 μm, orthogonal image 5 μm; (c, d) 40 μm, orthogonal image 5 μm; (e, f) 10 μm, orthogonal image 5 μm; (g) 20 μm, orthogonal image 5 μm. GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
Mentions: Further confocal analysis revealed that predifferentiated SVZ-hNSC grafts expressed GAD65/67 (Fig. 6a), as well as the intracellular CBPs CB (Fig. 6b), CR (Fig. 6c), and PV (Fig. 6d). In addition, predifferentiated SVZ-hNSCs were observed to express SYN that was mostly localized to the cell body of cells transplanted into the core infarct region (Fig. 6e), but was also additionally observed along the cytoskeleton of cells that were transplanted into sites outside of the infarct within the peri-infarct territory (Fig. 6f). Predifferentiated SVZ-hNSC grafts located within the peri-infarct territory appeared to extend long neurites revealed by HuNu and Tuj1 immunostaining (Fig. 6g). Grafts located within the core infarct were surrounded by the glial scar consisting of densely packed GFAP-positive astrocytes, with limited dispersion of SVZ-hNSCs in closest proximity to the scar (Fig. 7a–c). Parallel negative control experiments omitting primary antibodies depict a low level of autofluorescence within the core infarct (Fig. d–f).Fig. 6

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