使用主神经球文化研究纤毛
Summary
初级纤毛是在神经祖细胞增殖，神经细胞的分化，和成人神经元功能至关重要的。这里，我们描述研究ciliogenesis和信号蛋白使用初级神经球培养物在神经干/祖细胞和分化的神经元纤毛的贩卖的方法。
Abstract
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.
Introduction
初级纤毛是基于微管的动态亚细胞区室，其用作在胚胎神经元发育1,2期间协调细胞信号传导途径，包括音猬因子（Shh）途径感官天线，并且在成年神经元功能3条块亚细胞信号传导，4 。这些信号途径，如修补5的Shh受体的部件;该途径激活平滑（SMO）6;和Gpr161 7，孤儿G蛋白偶联受体（GPCR），所述Shh通路负调节，定位于纤毛以动态方式。多的GPCR据报道，本地化的纤毛神经元在大脑中7，8，9，10 </SUP>，11，12，13，14，15，16。在纤毛和纤毛生成的信号传导途径的缺陷影响多种组织和被统称为ciliopathies 17，18，19。所述ciliopathy疾病谱频繁包括神经发育缺陷，如颅面畸形20，21，22。另外，在丘脑神经元初级纤毛调节饱腹感的中心通路，和缺陷导致中心性肥胖23，在综合征ciliopathies如巴比Biedel综合征24镜像肥胖。此外，神经肽受体纤毛信号调节CENTR人饱腹感通路11，14。如在海马神经元生长抑素受体3腺苷酸环化酶III（ACIII）和GPCR的睫状定位导致新物体识别缺陷和记忆缺陷25,26和平行缺乏睫状完整性27。纤毛生成的信令的发展方面是紧密联系在一起组织动态平衡;特别是，纤毛是在小脑28,29从颗粒祖细胞而产生的Shh亚型髓母细胞瘤的进展重要。因此，纤毛胚胎，产后，和成人的神经元的发育和功能的过程中发挥重要作用。
神经干细胞（NSCs）驻留在脑室下区侧脑室（SVZ），海马的齿状回的颗粒下区，并且在哺乳动物中30，31，32下丘脑第三脑室的脑室区。神经干细胞是多能，具有自我更新的能力，是大脑发育和再生医学30重要。在SVZ最神经干细胞是静态和具有一个孤初级纤毛的是，在许多情况下，伸出到侧脑室33。通过各种受体的定位，诱导下游细胞应答，特别是有关的Shh，TGFβ，和受体酪氨酸激酶途径2，34，35，36中的初级纤毛信号。由于初级纤毛延伸进入侧脑室，假设初级纤毛检测在脑脊液（CSF），以激活神经干细胞的细胞因子37 </s了>。最近的研究表明Shh信号传导途径和初级纤毛是用于干细胞在修复的激活和再生多种组织中，包括嗅上皮，肺，肾和38，39，40，41是至关重要的。然而，通过该CSF的机制与神经干细胞进行通信，并且主是否纤毛参与是未知的。在贴壁培养神经干细胞的纤毛;本地化Shh通路部件，诸如SMO和Gpr161在纤毛;并响应嘘42。因此，神经干细胞可以作为研究Shh通路的重要模型系统，纤毛贩卖和神经元分化的途径。此外，从神经干细胞分化的神经元也可用于睫状贩卖测定。
神经球构成从neura的增殖所产生的自由浮动的细胞群的在特定的生长因子和非粘性表面43，44存在下生长升干/祖细胞。神经球来研究在正常发育和疾病31，45，46，47的神经干/祖细胞用作体外培养模型一样重要。在这里，我们描述了用于培养神经干/祖细胞和分化成神经元/神经胶质基于神经球的测定。我们特别强调信号元件为神经干/祖细胞和分化的神经元（ 图1）的纤毛的贩卖。相对于培养原代神经元，神经球都比较容易培养，是适合于多次传代和冻融循环，并且可以经历分化成神经元/神经胶质。重要的是，我们确定了神经球源性神经干/祖细胞和分化的神经元在纤毛培养和定位信号有关在这些隔室纤毛功能的分子。基于神经球培养方法可作为在神经干细胞和分化的神经元研究ciliogenesis和纤毛贩卖的理想模型系统。
Protocol
Representative Results
Discussion
在这里，我们描述了一个方法来生成和维护从成年小鼠SVZ神经球文化。是关于文化的几个相关要点如下。首先，球体的尺寸通常50之间 – 200微米。根据我们的经验，当一个神经球得到直径大于300微米，传代的最佳时机已经错过。这些较大的球体包含在核心死细胞。其次，由于神经球通常用于研究神经干/祖细胞，它使用EGF和碱性FGF（bFGF）的，以保持这些细胞的干性是很重要的。因此，因子诱导分?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Work in S.M.’s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).
Materials
| 12 mm round cover glass | Fisherbrand | 12-545-80 | |
| 24 well plate | Falcon | 353047 | |
| 4 % paraformaldehyde | Affymetrix | 19943 | |
| 50 ml tube | Falcon | 352098 | |
| 95 mm X 15 mm petri dish, slippable lid | Fisherbrand | FB0875714G | 10 cm dish |
| 70 µm cell strainer | Falcon | 352350 | |
| Alexa Fluor 488 Affinipure Donkey Anti-Rabbit IgG (H+L) | Jackson Immunoresearch | 711-545-152 | Donkey anti Rabbit, Alexa 488 secondary antibody |
| Arl13B, Clone N295B/66 | Neuromab | AB_11000053 | |
| B-27 Supplement (50X), serum free | ThermoFisher Scientific | 17504001 | B27 |
| Centrifuge | Thermo scientific | ST 40R | |
| Cryogenic vial | Corning | 430488 | |
| DAPI | Sigma | D9542-10MG | |
| Deoxyribonuclease I from bovine pancrease | Sigma | D5025-15KU | Dnase I |
| Dimethyl sulfoxide | Sigma | D8418-100ML | DMSO |
| Disposable Vinyl Specimen Molds | Sakura Tissue-Tek Cryomold | 4565 | 10 mm X 10 mm X 5 mm |
| Dulbecco's Phosphate Buffered Saline 10×, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture | Sigma | D1408-500ML | PBS |
| Dumont #5 Forceps | Fine science tools | 11254-20 | |
| FBS | Sigma | F9026-500ML | |
| Fluoromount-G solution | Southern Biotech | 0100-01 | mounting solution |
| GFAP | DAKO | Z0334 | |
| Goat anti Mouse IgG1 Secondary Antibody, Alexa Fluor 555 conjugate | ThermoFisher Scientific | A-21127 | Goat anti Mouse IgG1, Alexa 555 secondary antibody |
| Goat anti Mouse IgG2a Secondary Antibody, Alexa Fluor 555 conjugate | ThermoFisher Scientific | A-21137 | Goat anti Mouse IgG2a, Alexa 555 secondary antibody |
| Gpr161 | home made | N/A | |
| human bFGF | Sigma | F0291 | FGF |
| hemocytometer | Hausser Scientific | 0.100 mm deep | improved neubauer |
| Isothesia | Henry Schein | NDC 11695-0500-2 | Isofluorane |
| Laminin from Engelbreth-Holm-Swarm Sarcoma basement membrane | Sigma | L2020 | Laminin |
| L-Glutamine (200mM) | Sigma | G7513 | |
| Lipofectamine 3000 Transfection Reagent | ThermoFisher Scientific | L3000 | |
| Mr. Frosty | Nalgene | 5100-0036 | |
| N-2 supplement (100X) | ThermoFisher Scientific | 17502001 | N2 |
| Neurobasal medium | Gibco | 21103-049 | |
| Normal Donkey Serum | Jackson ImmunoResearch | 017-000-121 | |
| OCT compound | Sakura Tissue-Tek | 4583 | OCT |
| Penicillin-Streptomycin | Sigma | P4333-100ML | |
| Poly-L-lysine | Sigma | P4707 | |
| Recombinant human EGF protein, CF | R and D systems | 236-EG-200 | EGF |
| Scissor | Fine science tools | 14060-10 | |
| Superfrost plus microscope slide | Fisher scientific | 12-550-15 | slides |
| Triton X | Bio-Rad | 161-0407 | |
| Trypsin-EDTA solution (10X) | Sigma | T4174-100 | Trypsin |
| COSTAR 6 Well Plate, With Lid Flat Bottom Ultra-Low Attachment Surface Polystyrene, Sterile | Corning | 3471 | ultra-low binding 6 well plate |
| β-tubulin III | Covance | MMS-435P | TUJ1 |
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