Abstract
BackgroundNeural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it's estimated to be 2.5 to 3%.Methodology/Principal FindingsWe investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.Conclusions/SignificanceThese findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
Highlights
Cultured central nervous system (CNS) stem cells are endowed with capacity to selfrenew and differentiate into neurons, astrocytes and oligodendrocytes in predictable proportions [1,2,3,4,5]
We recently transplantated IhNSC-derived progenitors (IhNSC-P) near the hippocampal CA1 layer of adult rats injured by global transient ischemia to evaluate the integration and maturation of hNSC in a context mimicking the chronic impairment of neurological function following hypoxia-induced injuries, and documented their ability to engraft efficiently, to the point of establishing synaptic contacts with the host cells (Rota Nodari et al, submitted). Considering these findings, we have examined the effects of different oxygen concentrations (1%, 2.5%, 5% and 20% atmospheric oxygen) on the proliferation, differentiation and death of IhNSC in order to identify the optimal culture conditions of non-immortalized human Neural stem cells (NSCs) for NSC-mediated therapy of CNS injuries characterized by severe hypoxia-associated cell death that occurs in stroke and ischemia
We have shown that mild hypoxia (2.5– 5% O2), which better approximates the physiological setting, promotes survival of actively replicating IhNSCs as well as the yield of oligodendroglial and neuronal cells
Summary
Cultured CNS stem cells are endowed with capacity to selfrenew and differentiate into neurons, astrocytes and oligodendrocytes in predictable proportions [1,2,3,4,5] They have provided a useful tool to elucidate the pathways leading to generation of neurons and glia and to study the effects of different extrinsic factors on the commitment of neural stem cells (NSC) to form such cell lineages [6]. For these reasons the discovery, isolation and characterization of multipotent NSC from various locations within the mammalian brain represents a major recent advancement in neuroscience [7]. In the in the subventricular zone niche area, it’s estimated to be 2.5 to 3%
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