Abstract
IntroductionDespite 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.MethodsPre-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.ResultsBehavioral 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.ConclusionOur 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.
Highlights
Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits
In vitro characterization of cells Confocal analysis of immunolabeled undifferentiated subventricular zone (SVZ)-Human neural stem cell (hNSC) in vitro revealed formation of neurospheres that were colocalized with Human specific nuclear antigen (HuNu) and the undifferentiated cell marker SOX2 (98.9 ± 0.7 %) (Fig. 1a)
Predifferentiated SVZhNSCs labeled with HuNu were positive for β-III Tubulin (Tuj1) (92.3 ± 1.4 %) (Fig. 1g), gamma-aminobutyric acid (GABA) (90.1 ± 2.7 %) (Fig. 1h), Glutamate decarboxylase 65&67 (GAD) (Fig. 1i), and DCX (Fig. 1j)
Summary
Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. 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. Exogenous cell-based therapies to complement endogenous repair mechanisms are currently being trialed in humans following extensive meta-analysis of over 40 studies reporting significant improvements in function after cell transplantation in ischemic animal models [9,10,11]. Despite early reports of functional benefits in humans [12, 13], a cellular basis for neurological improvement still remains elusive [14]. Whilst neural cell replacement may be achieved, new research shows that neural stem cells (NSCs) can exert trophic effects through secretion of protein factors which induce change in the host tissue to promote functional improvements [15]. In addition to identifying how these cells work, it is important to isolate factors that influence stem cell survival and long-term integration within host tissue
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