Abstract
Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinson's disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1±0.5 and 17.1±0.4 (P<0.001); forebrain: 1.9±0.4 and 3.9±0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.
Highlights
Parkinson’s disease (PD) is an incurable neurodegenerative disorder affecting approximately 1% of the population over 60 years of age
To characterize and compare midbrain and forebrain Neural stem cells (NSCs) undergoing expansion, cells were cultured for 4 days in vitro (DIV) in medium containing the mitogens bFGF and EGF and immunostained for various markers
The content of dividing Ki67-immunoreactive (-ir) cells was found to be significantly higher for midbrain (8769% of total cells) compared to forebrain NSCs (5267% of total cells) (Fig.1 A,B)
Summary
Parkinson’s disease (PD) is an incurable neurodegenerative disorder affecting approximately 1% of the population over 60 years of age. A number of explorative studies using human fetal, ventral mesencephalic (VM) dopaminergic neurons have shown that intrastriatal transplantation may become an effective future treatment for patients with PD [2,3,4,5]. Neural stem cells (NSCs) are proliferative, multipotent cells that can be isolated from specific regions of the developing and mature central nervous system (CNS). Such cells may have significant advantages compared to human fetal VM tissue as they can be propagated to almost unlimited numbers of relatively homogenous cells in vitro and frozen without significant loss of cell viability. An efficient protocol for controlled generation of transplantable and functional dopaminergic neurons is still not available
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