Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway

Abstract: 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.