Shimada Issei S, Badgandi Hemant, Somatilaka Bandarigoda N, Mukhopadhyay Saikat
Department of Cell Biology, University of Texas Southwestern Medical Center;
Department of Cell Biology, University of Texas Southwestern Medical Center.
J Vis Exp. 2017 Apr 14(122):55315. doi: 10.3791/55315.
The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.
初级纤毛对于神经干细胞/祖细胞的增殖以及胚胎期、出生后和成年期的神经元分化至关重要。此外，大多数分化的神经元都拥有初级纤毛，其中包含信号受体，如G蛋白偶联受体，以及信号分子，如腺苷酸环化酶。初级纤毛决定了多种发育途径的活性，包括胚胎神经元发育过程中的音猬因子途径，并且在成年神经元功能中也起着促进亚细胞信号分隔的作用。不出所料，初级纤毛生物发生和功能的缺陷与大脑发育异常、中枢性肥胖以及学习和记忆缺陷有关。因此，在神经干细胞/祖细胞和分化神经元的背景下研究初级纤毛生物发生和纤毛运输势在必行。然而，原代神经元的培养方法需要相当多的专业知识，并且不适合冻融循环。在本方案中，我们讨论了使用原代神经球培养神经干细胞/祖细胞混合群体的方法。基于神经球的培养方法提供了研究原代神经干细胞/祖细胞的综合优势：易于多次传代和冻融循环、具有分化为神经元/神经胶质细胞的潜力以及可转染性。重要的是，我们确定神经球衍生的神经干细胞/祖细胞和分化的神经元在培养中具有纤毛，并在这些区室中定位与纤毛功能相关的信号分子。利用这些培养物，我们进一步描述了研究神经干细胞/祖细胞和分化神经元中纤毛发生和纤毛运输的方法。这些基于神经球的方法使我们能够在神经干细胞/祖细胞和分化神经元的背景下研究纤毛调节的细胞途径，包括G蛋白偶联受体和音猬因子信号传导。
