Dey Devanjan, Shrivastava Vadanya, Joshi Diksha, Singal Chitra Mohinder Singh, Tyagi Sagar, Bhat Muzaffer Ahmed, Jaiswal Paritosh, Sharma Jai Bhagwan, Palanichamy Jayanth Kumar, Sinha Subrata, Seth Pankaj, Sen Sudip
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
Department of Molecular and Cellular Neuroscience, National Brain Research Centre, Manesar, Haryana, India.
Mol Neurobiol. 2023 May;60(5):2910-2921. doi: 10.1007/s12035-023-03248-4. Epub 2023 Feb 7.
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 than 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 subventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression using immunocytochemistry and flow cytometry, respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 h, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4 × 44 K human array slides) highlighted that genes associated with neurogenesis were enriched upon exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed the involvement of the WNT signaling pathway. Microarray analyses were validated using neuronal and glial lineage commitment markers, namely, NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2, and NKX2.2, using qPCR (n = 9). DCX, ASCL1, NGN1, and GFAP protein expression was analyzed by Western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers upon 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 upon exposure to hypoxia.
人类胎儿大脑中存在的胎儿神经干细胞（FNSCs）可分化为神经元和神经胶质谱系的细胞。发育中的胎儿所接触的氧气浓度低于成年人，这种生理性缺氧可能会影响FNSCs的生长和分化。本研究旨在评估缺氧对从流产胎儿脑室下区分离的人类FNSCs（n = 5）分化潜能的影响。通过免疫细胞化学和流式细胞术分别对FNSCs进行分离、扩增，并根据巢蛋白（Nestin）和性别决定区Y框蛋白2（Sox2）的表达进行鉴定。将这些FNSCs暴露于20%氧气（常氧）和0.2%氧气（缺氧）浓度下48小时，并对缺氧暴露情况（n = 5）进行验证。对暴露于缺氧环境的FNSCs进行全转录组分析（Genespring GX13，使用安捷伦4×44K人类阵列玻片），结果突出显示，与神经发生相关的基因在缺氧暴露后富集。对这些富集基因进行通路分析（使用Metacore）表明，WNT信号通路参与其中。使用qPCR（n = 9），以神经元和神经胶质谱系定向标志物，即神经发生蛋白1（NEUROG1）、神经发生蛋白2（NEUROG2）、无翅型MMTV整合位点家族成员1（ASCL1）、双皮质素（DCX）、胶质纤维酸性蛋白（GFAP）、少突胶质细胞转录因子2（OLIG2）和NK2同源盒2（NKX2.2），对微阵列分析进行验证。通过蛋白质免疫印迹法（n = 3）分析DCX、ASCL1、神经发生蛋白1（NGN1）和GFAP的蛋白表达。这表明缺氧暴露后神经元定向标志物上调，而星形胶质细胞和少突胶质细胞谱系定向标志物未观察到变化。通过qPCR（n = 9）检测发现WNT信号通路下游靶点TCF4和ID2的表达增加，通过蛋白质免疫印迹法（n = 3）检测发现β-连环蛋白（CTNNB1）和ID2的蛋白表达增加，表明其参与介导缺氧暴露后的神经元分化。
