Stevanato Lara, Hicks Caroline, Sinden John D
ReNeuron;
ReNeuron.
J Vis Exp. 2015 Apr 12(98):52410. doi: 10.3791/52410.
Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER(TAM)construct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3'UTRs by dual reporter plasmid transfection and dual luciferase assay.
神经干细胞（NSCs）能够自我更新，并在特定的局部微环境下分化为神经元、星形胶质细胞和少突胶质细胞。在此，我们展示了一套用于对一种克隆性人类神经干细胞（hNSC）系进行三维（3D）分化和miRNA分析的方法，该细胞系目前正处于中风残疾的临床试验中（NCT01151124和NCT02117635，Clinicaltrials.gov）。hNSCs源自经伦理批准的孕早期人类胎儿皮质，并通过逆转录病毒整合单拷贝的c-mycER(TAM)构建体进行条件性永生化。我们描述了如何测量在3D支架上分化的hNSCs的轴突生长过程，以及如何使用PCR阵列定量miRNA表达的相关变化。此外，我们举例说明了旨在选择miRNA假定靶点的计算分析。SOX5和NR4A3被确定为在分化的hNSC中选定的显著下调miRNA的合适假定靶点。通过双报告质粒转染和双荧光素酶测定对SOX5和NR4A3 3'UTR进行miRNA靶点验证。
