Fetal neural stem cells (FNSCs) present in the human fetal brain, differentiate into cells of neuronal and glial lineages. The developing fetus is exposed to lower oxygen concentrations compared to adults and this physiological hypoxia may in uence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the subventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and owcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were enriched on exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed that the involvement of WNT signaling pathway. Microarray analyses was validated using neuronal and glial lineage commitment markers, namely NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2 and NKX2.2 using qPCR (n = 9). DCX and GFAP protein expression were analysed by western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n = 9), and increased protein expression of CTNNB1 (β-catenin) and ID2 by western blot (n = 3), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.
Hypoxic ischemic injury to the fetal and neonatal brain is a leading cause of death and disability worldwide. Although animal and culture studies suggest that glutamate excitotoxicity is a primary contributor to neuronal death following hypoxia, the molecular mechanisms, and roles of various neural cells in the development of glutamate excitotoxicity in humans, is not fully understood. In this study, we developed a culture model of human fetal neural stem cell (FNSC)-derived astrocytes and examined their glutamate uptake in response to hypoxia. We isolated, established, and characterized cultures of FNSCs from aborted fetal brains and differentiated them into astrocytes, characterized by increased expression of the astrocyte markers glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1) and EAAT2, and decreased expression of neural stem cell marker Nestin. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2%, respectively). Interestingly, no change was observed in the expression of the glutamate transporter EAAT2 or glutamate uptake by astrocytes, even after exposure to severe hypoxia for 48 h. These results together suggest that human FNSC-derived astrocytes can maintain glutamate uptake after hypoxic injury and thus provide evidence for the possible neuroprotective role of astrocytes in hypoxic conditions.
Fetal neural stem cells (FNSCs) present in the human fetal brain, differentiate into cells of neuronal and glial lineages. The developing fetus is exposed to lower oxygen concentrations compared to adults and this physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were enriched on exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed that the involvement of WNT signaling pathway. Microarray analyses was validated using neuronal and glial lineage commitment markers, namely NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2 and NKX2.2 using qPCR (n = 9). DCX and GFAP protein expression were analysed by western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n = 9), and increased protein expression of CTNNB1 (β-catenin) and ID2 by western blot (n = 3), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.
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