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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 vitro. Immunofluorescent confocal images of undifferentiated SVZ-hNSCs in culture expressed HuNu (red) and SOX2 (green) (a), and Ki67 (red) and Nestin (green) (b), with colocalization giving a yellow appearance. Undifferentiated SVZ-hNSCs immunopositive for HuNu (red) (c–f) in culture were not observed to express Tuj1 (green) (c), GABA (green) (d), GAD (green) e, or DCX (green) f. Predifferentiated cells cultured for 7 days demonstrated immunoreactivity for HuNu (red) g–m and colabeled with Tuj1 (green) (g), GABA (green) (h), GAD (green) (i), and DCX (green) (j). Predifferentiated cells HuNu (red) cultured for 7 days demonstrated little immunoreactivity for Ki67 (green) (k), Nestin (green) (l), or SOX2 (green) (m). Scale bar: (a–f) 100 μm, (g–m) 50 μm. DCX doublecortin, GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
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Fig1: Undifferentiated hNSCs and predifferentiated cell phenotypes in vitro. Immunofluorescent confocal images of undifferentiated SVZ-hNSCs in culture expressed HuNu (red) and SOX2 (green) (a), and Ki67 (red) and Nestin (green) (b), with colocalization giving a yellow appearance. Undifferentiated SVZ-hNSCs immunopositive for HuNu (red) (c–f) in culture were not observed to express Tuj1 (green) (c), GABA (green) (d), GAD (green) e, or DCX (green) f. Predifferentiated cells cultured for 7 days demonstrated immunoreactivity for HuNu (red) g–m and colabeled with Tuj1 (green) (g), GABA (green) (h), GAD (green) (i), and DCX (green) (j). Predifferentiated cells HuNu (red) cultured for 7 days demonstrated little immunoreactivity for Ki67 (green) (k), Nestin (green) (l), or SOX2 (green) (m). Scale bar: (a–f) 100 μm, (g–m) 50 μm. DCX doublecortin, GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin

Mentions: Confocal analysis of immunolabeled undifferentiated SVZ-hNSCs in vitro revealed formation of neurospheres that were colocalized with HuNu and the undifferentiated cell marker SOX2 (98.9 ± 0.7 %) (Fig. 1a). These cells also colocalized with proliferation marker Ki67 (59.5 ± 3.1 %) and NSC marker Nestin (95.1 ± 0.7 %) (Fig. 1b), but did not express early neuronal marker Tuj1, inhibitory neurotransmitter GABA, GABA-producing enzyme GAD, or immature neuronal marker DCX (Fig. 1c–f). In contrast, predifferentiated SVZ-hNSCs labeled with HuNu were positive for Tuj1 (92.3 ± 1.4 %) (Fig. 1g), GABA (90.1 ± 2.7 %) (Fig. 1h), GAD (Fig. 1i), and DCX (Fig. 1j). Very few predifferentiated cells expressed Ki67 (9.6 ± 2.5 %) (Fig. 1k) or Nestin (28.5 ± 2.6 %, Fig. 1l) and SOX2 (12.1 ± 1.7 %, Fig. 1m). No expression of GFAP, SYN, or the intracellular calcium binding proteins (CBPs) CB, CR, or PV was detected in vitro.Fig. 1


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 vitro. Immunofluorescent confocal images of undifferentiated SVZ-hNSCs in culture expressed HuNu (red) and SOX2 (green) (a), and Ki67 (red) and Nestin (green) (b), with colocalization giving a yellow appearance. Undifferentiated SVZ-hNSCs immunopositive for HuNu (red) (c–f) in culture were not observed to express Tuj1 (green) (c), GABA (green) (d), GAD (green) e, or DCX (green) f. Predifferentiated cells cultured for 7 days demonstrated immunoreactivity for HuNu (red) g–m and colabeled with Tuj1 (green) (g), GABA (green) (h), GAD (green) (i), and DCX (green) (j). Predifferentiated cells HuNu (red) cultured for 7 days demonstrated little immunoreactivity for Ki67 (green) (k), Nestin (green) (l), or SOX2 (green) (m). Scale bar: (a–f) 100 μm, (g–m) 50 μm. DCX doublecortin, GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
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Fig1: Undifferentiated hNSCs and predifferentiated cell phenotypes in vitro. Immunofluorescent confocal images of undifferentiated SVZ-hNSCs in culture expressed HuNu (red) and SOX2 (green) (a), and Ki67 (red) and Nestin (green) (b), with colocalization giving a yellow appearance. Undifferentiated SVZ-hNSCs immunopositive for HuNu (red) (c–f) in culture were not observed to express Tuj1 (green) (c), GABA (green) (d), GAD (green) e, or DCX (green) f. Predifferentiated cells cultured for 7 days demonstrated immunoreactivity for HuNu (red) g–m and colabeled with Tuj1 (green) (g), GABA (green) (h), GAD (green) (i), and DCX (green) (j). Predifferentiated cells HuNu (red) cultured for 7 days demonstrated little immunoreactivity for Ki67 (green) (k), Nestin (green) (l), or SOX2 (green) (m). Scale bar: (a–f) 100 μm, (g–m) 50 μm. DCX doublecortin, GABA gamma-aminobutyric acid, GAD glutamate decarboxylase 65&67, HuNu human specific nuclear antigen, Tuj1 β-III tubulin
Mentions: Confocal analysis of immunolabeled undifferentiated SVZ-hNSCs in vitro revealed formation of neurospheres that were colocalized with HuNu and the undifferentiated cell marker SOX2 (98.9 ± 0.7 %) (Fig. 1a). These cells also colocalized with proliferation marker Ki67 (59.5 ± 3.1 %) and NSC marker Nestin (95.1 ± 0.7 %) (Fig. 1b), but did not express early neuronal marker Tuj1, inhibitory neurotransmitter GABA, GABA-producing enzyme GAD, or immature neuronal marker DCX (Fig. 1c–f). In contrast, predifferentiated SVZ-hNSCs labeled with HuNu were positive for Tuj1 (92.3 ± 1.4 %) (Fig. 1g), GABA (90.1 ± 2.7 %) (Fig. 1h), GAD (Fig. 1i), and DCX (Fig. 1j). Very few predifferentiated cells expressed Ki67 (9.6 ± 2.5 %) (Fig. 1k) or Nestin (28.5 ± 2.6 %, Fig. 1l) and SOX2 (12.1 ± 1.7 %, Fig. 1m). No expression of GFAP, SYN, or the intracellular calcium binding proteins (CBPs) CB, CR, or PV was detected in vitro.Fig. 1

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