Stable STIM1 knockdown in human neural precursors derived from hESC line, H9

Ca2 + signaling plays a significant role in development of the vertebrate nervous system where it regulates neurite growth as well as synapse and neurotransmitter specification (Rosenberg and Spitzer, 2011). Elucidating the role of Ca2 + signaling in neuronal development has been largely restricted to either small animal models or primary cultures. Here we derived human neural precursor cells (NPCs) from human embryonic stem cells to understand the functional significance of a less understood arm of calcium signaling, Store-operated Ca2+ entry or SOCE, in neuronal development. Human NPCs exhibited robust SOCE, which was significantly attenuated by expression of a stable shRNA-miR targeted towards the SOCE molecule STIM1. Along with the plasma membrane channel Orai, STIM is an essential component of SOCE in many cell types where it regulates gene expression. Therefore, we measured global gene expression in human NPCs with and without STIM1 knockdown. Interestingly, pathways down-regulated through STIM1 knockdown were related to cell proliferation and DNA replication processes whereas post-synaptic signaling was identified as an up-regulated process. To understand the functional significance of these gene expression changes we measured the self-renewal capacity of NPCs with STIM1 knockdown. These demonstrated significantly reduced neurosphere size and number as compared to control cells. Moreover, spontaneous differentiation towards the neuronal lineage was enhanced. These findings demonstrate that STIM1 mediated SOCE in human NPCs regulates gene expression changes, that in vivo are likely to physiologically modulate the self-renewal and differentiation of NPCs. STIM1 knockdown in human embryonis stem cell derived neural progenitor cells (NPCs) by using ShERWOOD-UltramiR short hairpin RNA (shRNA). Hence we designed our RNA seq in such a way that we can compare the differential gene expression data using STIM1 Knockdown (KD) as test group, shRNA non-targetted control (NTC) and wild type NPC (WT) as control groups. Biological triplicates were performed for each sample consisting of RNA isolated from wild type NPCs.

钙离子信号（Ca²+ signaling）在脊椎动物神经系统发育中发挥重要作用，其可调控神经突生长、突触形成与神经递质表型特化（Rosenberg及Spitzer，2011）。此前针对钙离子信号在神经元发育中功能的解析，大多局限于小型动物模型或原代培养体系。本研究通过人类胚胎干细胞诱导获得人类神经前体细胞（human neural precursor cells, NPCs），以期阐明钙信号通路中较未被充分研究的分支——钙池调控性钙离子内流（Store-operated Ca²+ entry, SOCE）在神经元发育中的功能意义。人类神经前体细胞可检测到显著的SOCE活性，而通过靶向SOCE核心分子基质相互作用分子1（STIM1）的稳定表达短发卡RNA-miR（shRNA-miR），可显著削弱该活性。与质膜通道Orai（Orai）共同作用时，STIM是众多细胞类型中SOCE的必需组分，可调控基因表达。因此，我们对STIM1敲低与未敲低的人类神经前体细胞进行了全局基因表达分析。有趣的是，STIM1敲低后下调的通路主要与细胞增殖及DNA复制过程相关，而上调的通路则被鉴定为突触后信号通路。为解析这些基因表达变化的功能意义，我们检测了STIM1敲低后神经前体细胞的自我更新能力。结果显示，与对照细胞相比，STIM1敲低组的神经球大小与数量均显著降低。此外，向神经元谱系的自发分化过程得到增强。上述研究结果表明，人类神经前体细胞中STIM1介导的SOCE可调控基因表达变化，这些变化在体内可能生理性调控神经前体细胞的自我更新与分化。本研究通过ShERWOOD-UltramiR短发卡RNA（shRNA）对人类胚胎干细胞来源的神经前体细胞（NPCs）进行了STIM1敲低。因此，我们设计了RNA测序（RNA-seq）实验方案，可将STIM1敲低（STIM1 KD）组作为实验组，以非靶向shRNA对照（NTC）与野生型神经前体细胞（WT）作为对照组，开展差异基因表达数据的比较分析。每个由野生型神经前体细胞分离得到RNA的样本，均设置了三次生物学重复。