Article

Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
PLoS ONE (Impact Factor: 3.53). 01/2010; 5(1):e8575. DOI: 10.1371/journal.pone.0008575
Source: PubMed

ABSTRACT Neural 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%.
We 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 beta-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.
These 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.

0 Followers
 · 
190 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hypoxia is an important factor in regulation of cell behavior both under physiological and pathological conditions. The mechanisms of hypoxia-induced cell death have not been completely elucidated yet. It is well known that Ca2+ is critically related to cell survival. Hypoxia-inducible factor-1α (HIF-1α) is a core regulatory factor during hypoxia, and L-type voltage-dependent Ca2+ channels (L-VDCCs) have been reported to play a critical role in cell survival. This study was conducted to explore the relationship between L-VDCC expression and HIF-1α regulation in PC12 cells under hypoxia. PC12 cells were treated at 20 or 3 % O2 to observe its proliferation and the intracellular calcium concentration. Then, we detected the protein expression of HIF-1α and L-VDCCs subtypes, Cav1.2 and Cav1.3. At last, to verify the relationship between HIF-1α and Cav1.2 and Cav1.3, we got the expression of Cav1.2 and Cav1.3 with Western blot and luciferase report gene assays after PC12 cells were treated by echinomycin, which is an HIF-1α inhibitor. Compared with 20 % O2 (normoxia), 3 % O2 (hypoxia) inhibited cell proliferation, increased the intracellular calcium concentration, and induced protein expression of HIF-1α. The protein expression of two L-VDCCs subtypes expressed in the nervous system, Cav1.2 and Cav1.3, was upregulated by hypoxia and reduced dose dependently by treatment with echinomycin, a HIF-1α inhibitor. Luciferase report gene assays showed that the expression of Cav1.2 and Cav1.3 genes was augmented under 3 % O2. However, echinomycin only slightly and dose dependently decreased expression of the Cav1.2 gene, but not that of the Cav1.3 gene. These data indicated that Cav1.2 might be regulated by HIF-1α as one of its downstream target genes and involved in regulation of PC12 cells death under hypoxia.
    Cell Stress and Chaperones 02/2015; 20(3). DOI:10.1007/s12192-015-0575-2 · 2.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state. © 2015 S. Karger AG, Basel.
    Cells Tissues Organs 02/2015; DOI:10.1159/000371342 · 2.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human adipose tissue-derived multilineage progenitor cells (hADMPCs) are attractive for cell therapy and tissue engineering because of their multipotency and ease of isolation without serial ethical issues. However, their limited in vitro lifespan in culture systems hinders their therapeutic application. Some somatic stem cells including hADMPCs are known to be localized in hypoxic regions; thus, hypoxia may be beneficial for ex vivo culture of these stem cells. These cells exhibit a high level of glycolytic metabolism in presence of high oxygen levels and further increase their glycolysis rate under hypoxia. However, the physiological role of glycolytic activation and its regulatory mechanisms are still incompletely understood. Here we show that Notch signaling is required for glycolysis regulation under hypoxic conditions. Our results demonstrate that 5% O2 dramatically increased the glycolysis rate, improved the proliferation efficiency, prevented senescence, and maintained the multipotency of hADMPCs. Intriguingly, these effects were not mediated by hypoxia-inducible factor (HIF), but rather by the Notch signaling pathway. 5% O2 significantly increased the level of activated Notch1 and expression of its downstream gene, HES1. Furthermore, 5% O2 markedly increased glucose consumption and lactate production of hADMPCs, which decreased back to normoxic levels upon treatment with a γ-secretase inhibitor. We also found that HES1 was involved in induction of GLUT3, TPI, and PGK1 in addition to reduction of TIGAR and SCO2 expression. These results clearly suggest that Notch signaling regulates glycolysis under hypoxic conditions and thus likely affects the cell lifespan via glycolysis.
    Stem Cells and Development 05/2014; DOI:10.1089/scd.2013.0642 · 4.20 Impact Factor