Abstract
PurposeTo investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.MethodsCerebral cortical cells from neonatal Sprague–Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named ‘hypoxic conditioned medium’ (HCM) and ‘normoxic conditioned medium’ (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.ResultsThe protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by LY294002 and SP600125, with LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by LY294002 and SP600125.Conclusions4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.
Highlights
Neural stem cells (NSCs) are multipotent cells that have the capacity to self-renew and differentiate into neurons, astrocytes and oligodendrocytes [1]
We selected 4% O2 and 1% O2 to stimulate SD rat cerebral cortical cells in vitro, and our findings showed that both hypoxic conditions promoted cerebral cortical cells to express VEGF and BDNF mRNA, and to induce cells to secrete VEGF and BDNF into the conditioned media as compared to the normoxic stimulations
There is evidence to suggest that stroke or traumatic brain injury/ischemia could induce the expression of trophic factors and cytokines from NSCs, endothelial cells or other cells in vivo [13]
Summary
Neural stem cells (NSCs) are multipotent cells that have the capacity to self-renew and differentiate into neurons, astrocytes and oligodendrocytes [1]. They are present mainly in the developing and adult central nervous system (CNS) of mammals, including humans, where they are found in two brain regions: the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus [2]. The discovery of NSCs led to the hope that stem cell transplantation could be used to treat neurodegenerative diseases. It has been demonstrated that most NSCs are in a quiescent state under physiological conditions, but that pathological conditions, such as stroke or ischemia, could affect their proliferation and differentiation. Hypoxia affects critical cellular processes, such as survival, proliferation, apoptosis, extracellular matrix secretion, the expression of neurotrophic factors, and neuronal differentiation [3,4,5,6]
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