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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

Undifferentiated hNSCs and predifferentiated cell phenotypes in vivo. Confocal immunofluorescent photomicrographs of undifferentiated hNSCs at 28 days post transplant within cortical border regions with orthogonal reconstructions. Undifferentiated hNSCs maintained expression of HuNu (red) and colabeled mainly with GFAP (a), Nestin (b), and Ki67 (c), with little expression of GABA (d) and Tuj1 (e) (green). Immunofluorescent confocal images of predifferentiated cells 28 days post transplant within the stroke-damaged cortex maintained expression of HuNu (red) colabeled with Tuj1 (f), GABA (g), Nestin (h), and Ki67 (i) (green), with lack of colocalization with GFAP (green) (j). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. HuNu immunoreactivity of undifferentiated hNSCs was observed completely surrounded by GFAP and Nestin, while HuNu immunoreactivity of predifferentiated cells was observed completely surrounded by Tuj1 and GABA. Scale bar: (a–e) 50 μm, orthogonal images 5 μm; (f–j) 20 μm, orthogonal images 5 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
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Fig4: Undifferentiated hNSCs and predifferentiated cell phenotypes in vivo. Confocal immunofluorescent photomicrographs of undifferentiated hNSCs at 28 days post transplant within cortical border regions with orthogonal reconstructions. Undifferentiated hNSCs maintained expression of HuNu (red) and colabeled mainly with GFAP (a), Nestin (b), and Ki67 (c), with little expression of GABA (d) and Tuj1 (e) (green). Immunofluorescent confocal images of predifferentiated cells 28 days post transplant within the stroke-damaged cortex maintained expression of HuNu (red) colabeled with Tuj1 (f), GABA (g), Nestin (h), and Ki67 (i) (green), with lack of colocalization with GFAP (green) (j). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. HuNu immunoreactivity of undifferentiated hNSCs was observed completely surrounded by GFAP and Nestin, while HuNu immunoreactivity of predifferentiated cells was observed completely surrounded by Tuj1 and GABA. Scale bar: (a–e) 50 μm, orthogonal images 5 μm; (f–j) 20 μm, orthogonal images 5 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, Tuj1 β-III tubulin

Mentions: Confocal image analysis 28 days post transplant revealed undifferentiated SVZ-hNSCs mainly expressed markers for GFAP and Nestin (Fig. 4a, b), with many cells also positive for Ki67 (Fig. 4c). Some undifferentiated SVZ-hNSCs were also found to express GABA and Tuj1 (Fig. 4d, e). In contrast, predifferentiated cells mainly expressed Tuj1 and GABA (Fig. 4f, g) with little colabeling with Nestin and Ki67 (Fig. 4h, i) and a distinct lack of GFAP expression (Fig. 4j).Fig. 4


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)

Undifferentiated hNSCs and predifferentiated cell phenotypes in vivo. Confocal immunofluorescent photomicrographs of undifferentiated hNSCs at 28 days post transplant within cortical border regions with orthogonal reconstructions. Undifferentiated hNSCs maintained expression of HuNu (red) and colabeled mainly with GFAP (a), Nestin (b), and Ki67 (c), with little expression of GABA (d) and Tuj1 (e) (green). Immunofluorescent confocal images of predifferentiated cells 28 days post transplant within the stroke-damaged cortex maintained expression of HuNu (red) colabeled with Tuj1 (f), GABA (g), Nestin (h), and Ki67 (i) (green), with lack of colocalization with GFAP (green) (j). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. HuNu immunoreactivity of undifferentiated hNSCs was observed completely surrounded by GFAP and Nestin, while HuNu immunoreactivity of predifferentiated cells was observed completely surrounded by Tuj1 and GABA. Scale bar: (a–e) 50 μm, orthogonal images 5 μm; (f–j) 20 μm, orthogonal images 5 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Undifferentiated hNSCs and predifferentiated cell phenotypes in vivo. Confocal immunofluorescent photomicrographs of undifferentiated hNSCs at 28 days post transplant within cortical border regions with orthogonal reconstructions. Undifferentiated hNSCs maintained expression of HuNu (red) and colabeled mainly with GFAP (a), Nestin (b), and Ki67 (c), with little expression of GABA (d) and Tuj1 (e) (green). Immunofluorescent confocal images of predifferentiated cells 28 days post transplant within the stroke-damaged cortex maintained expression of HuNu (red) colabeled with Tuj1 (f), GABA (g), Nestin (h), and Ki67 (i) (green), with lack of colocalization with GFAP (green) (j). Orthogonal reconstructions from confocal z-series are presented as viewed in x–z (top) and y–z (right) planes. HuNu immunoreactivity of undifferentiated hNSCs was observed completely surrounded by GFAP and Nestin, while HuNu immunoreactivity of predifferentiated cells was observed completely surrounded by Tuj1 and GABA. Scale bar: (a–e) 50 μm, orthogonal images 5 μm; (f–j) 20 μm, orthogonal images 5 μm. GABA gamma-aminobutyric acid, GFAP glial fibrillary acidic protein, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
Mentions: Confocal image analysis 28 days post transplant revealed undifferentiated SVZ-hNSCs mainly expressed markers for GFAP and Nestin (Fig. 4a, b), with many cells also positive for Ki67 (Fig. 4c). Some undifferentiated SVZ-hNSCs were also found to express GABA and Tuj1 (Fig. 4d, e). In contrast, predifferentiated cells mainly expressed Tuj1 and GABA (Fig. 4f, g) with little colabeling with Nestin and Ki67 (Fig. 4h, i) and a distinct lack of GFAP expression (Fig. 4j).Fig. 4

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