这项工作得到了NIH（1R35GM133496-01）对Z. Gao的资助。我们要感谢Ryan Hobbs博士在切片方面的帮助。我们感谢宾夕法尼亚州立大学医学院的核心设施，包括基因组科学和生物信息学，高级光学显微镜成像和流式细胞术。我们还感谢 Yuka Imamura 博士在 RNA-seq 分析方面的帮助。

1. 微微微网络培养
<ol><li>用0.1%明胶涂覆10厘米组织培养处理的平板，让明胶凝固至少15-30分钟，然后再吸出。</li>
	<li>在预热的mESC培养基（Dulbecco的改性鹰培养基（DMEM）中培养15%胎牛血清（FBS），非必需氨基酸，β巯基乙醇，L-谷氨酰胺，青霉素/链霉素，丙酮酸钠，LIF，PD0325901（PD）和Chir99021（CH））中培养mESCs之前一天，种子γ辐照小鼠胚胎成纤维细胞（MEFs）。</li>
	<li>在培养E14细胞之前，允许γ辐照的MEF沉降并附着在板表面。</li>
	<li>在37°C水浴中解冻E14ESCs，并用温暖的mESC培养基将细胞快速转移到15cm锥形管中。以200×g沉淀细胞3分钟并除去上清液。</li>
	<li>将细胞重悬于10mL mESC培养基中，并将细胞接种在含有先前接种的γ辐照MEF的培养板上。将细胞培养物在37°C培养箱中孵育5%CO2。</li>
	<li>对于培养传代，吸出培养基并用无菌的1x PBS洗涤板。加入足够的0.05%胰蛋白酶覆盖板表面，并在37°C下孵育3分钟。</li>
	<li>用mESC培养基和移液器中和胰蛋白酶以产生单细胞悬浮液。将细胞以200×g离心3分钟并除去上清液。</li>
	<li>用血细胞计数器或细胞计数器计数细胞，并在10cm培养板中接种约5.0×105 个细胞。</li>
	<li>将细胞重悬于10mL mESC培养基中，将细胞接种在明胶包被的组织培养板上，并如前所述孵育培养物。 注意:建议每2天传代一次mESCs，以防止细胞在其菌落中分化。培养基中的酚红仅起到pH指示剂的作用，根据细胞密度，它可以在2天内变黄（酸性更强）。因此，可能需要每天更换介质。γ辐照的MEF最终将在几次通过后死亡。</li>
</ol>2. EB、鼻咽癌和神经元分化
<ol><li>执行前面提到的培养传代方案并计数细胞（步骤1.7-1.10）。</li>
	<li>悬挂下降方法（第 0 天）<ol>
<li>对于10 cm细胞培养板，计数大约2.5×104 个细胞，其中5.0×102 个细胞将悬浮在20μL分化培养基中（DMEM与15%FBS，非必需氨基酸，β-巯基乙醇，L-谷氨酰胺，青霉素/链霉素和丙酮酸钠）。大约50个含有细胞的20μL液滴可以接种在一个10cm板上。</li>
		<li>等分适当数量的细胞，然后将细胞以200×g离心3分钟并除去上清液。</li>
		<li>将细胞重悬于适当体积的分化培养基中，使细胞密度为每20μL5.0×102 个细胞（例如，在1mL分化培养基中加入2.5×104 个细胞）。</li>
		<li>使用微量移液管或中继器移液器，将20μL细胞悬浮液滴置于组织培养板的盖子上。确保液滴彼此之间不太靠近，以防止它们合并。 注意:液滴可以接种在组织培养处理的附着板上或悬浮板上，因为它们将放置在盖子上而不是板本身。对于更可行和无菌的方法，将液滴放在附着的组织培养板上，并将它们转移到悬浮板上，如下所述。</li>
		<li>用 5–10 mL 1x PBS 填充盘子，然后小心地将盖子放回盘子上。在37°C培养箱中孵育培养物。 注意:将PBS添加到培养板中以防止液滴干燥。</li>
	</ol></li>
	<li>在第 2天，使用微量移液管从盖子收集液滴，并将它们放入充满10 mL分化培养基的10cm细胞培养悬浮板中。在培养箱中低速摇晃的轨道振荡器上孵育培养物。</li>
	<li>在第4天，为了收获EB，收集细胞，以200×g离心3分钟，然后除去上清液。 注意:EB也可以根据后续实验的要求用1x PBS洗涤。</li>
	<li>为了继续该过程并诱导EB分化为神经祖细胞（NPC），用5μM视黄酸（RA）制备分化培养基。</li>
	<li>通过以100 x g 沉淀EB3分钟来去除旧培养基，或者在吸出旧培养基之前让EB沉降。将含有5μM RA的10mL分化培养基加入培养板中。</li>
	<li>在第 6天，通过如上所述倾斜板并移出培养基，用含有5μM RA的新鲜培养基替换至少一半的培养基。 注意:建议在第5天和第7天用含有RA的新鲜培养基替换至少一半的培养基。注意酚红指示剂;如果它变成淡黄色，最好更换所有的培养基。</li>
	<li>在第8天，通过收集细胞，以200×g离心3分钟并除去上清液来收获NPC。 注意:NPC也可以根据后续实验的需要用1x PBS清洗。如果需要，NPC可以被冷冻并再次解冻，以便以后进行培养和分析。如果要培养NPC，accutase也可以用作胰蛋白酶的替代品。</li>
	<li>为了继续该过程并将NPC分化为神经元，通过离心将NPC收集在15mL锥形管中，用胰蛋白酶将其解离并在37°C下孵育3分钟。移液NPC以确保所有NPC聚集体解离并用培养基中和胰蛋白酶。</li>
	<li>用40μm尼龙细胞过滤器过滤细胞并计数细胞，然后将其以1.5 x 105/ cm2 的密度接种在N2培养基（DMEM / F12培养基+ 3mg / mL葡萄糖+ 3mg / mL富脂质牛血清白蛋白（LBSA）+ 1:100 N2补充剂+ 10 ng / mL bFGF + 50 U / mL笔/ 链球菌+ 1 mM L谷氨酰胺）中，用于随后的PCR和蛋白质印迹实验;或在组织培养室上进行免疫荧光实验。</li>
	<li>在第9天，用新鲜的N2培养基替换旧的培养基。</li>
	<li>在第 10天，将N2培养基与N2 / B27培养基（50%DMEM / F12和50%神经基础，3mg / mL LBA，1:200 N2补充剂，1:100 B27补充剂，50 U / mL笔/链球菌和1 mM L-谷氨酰胺）切换。</li>
	<li>在第11-12天，神经元的收获如下。用1x PBS洗涤细胞，加入胰蛋白酶，并在37°C培养箱中孵育3分钟，然后用培养基中和胰蛋白酶，并以200×g离心3分钟。 </li>
</ol>3. mESC和分化细胞的表征
<ol><li>碱性磷酸酶 （AP） 测定 
	<ol>
<li>使用试剂盒评估碱性磷酸酶活性（见 材料表）。</li>
		<li>从培养皿中取出培养基，并用1x PBS洗涤ESC。</li>
		<li>将1mL与试剂盒一起提供的固定溶液（由甲醛和甲醇组成）加入板中，并在室温下孵育2-5分钟。 注意:在修复解决方案中过度孵育可能会危及 AP 活动。</li>
		<li>取出固定溶液，用1x PBS洗涤ESC，并在板中留下一定量的PBS。 注意:在 PBS 中保持 ESC 湿润，以免影响 AP 活动。</li>
		<li>通过将 A、B 和 C 基底溶液以 1:1:1 的比例混合来制备 AP 溶液。首先混合A和B溶液，并在室温下孵育混合物2分钟，然后加入C溶液。</li>
		<li>删除 1 个 PBS 并添加之前准备的 AP 解决方案。</li>
		<li>通过用铝箔包裹培养板或在黑暗的房间里执行步骤3.5，在黑暗中孵育ESCs约15分钟。</li>
		<li>监测反应并在溶液变亮时除去反应溶液，以避免非特异性染色。</li>
		<li>用 1 倍 PBS 清洗 ESC 两次。</li>
		<li>通过用1x PBS或安装介质覆盖ESC来防止样品干燥。 注:AP 表达时会出现红色或紫色污渍。该板可以储存在4°C的冰箱中。</li>
	</ol></li>
	<li>RT-qPCR 
	<ol>
<li>通过分别对ESC和EB和NPC执行步骤1.6-1.7和2.4，在不同阶段收集细胞。</li>
		<li>使用RNA，DNA和蛋白质提取溶液分离RNA（见 材料表）。</li>
		<li>使用逆转录酶试剂盒（见 材料表）生成cDNA，并遵循制造商手册。</li>
	</ol></li>
	<li>固定和嵌入 
	<ol>
<li>如上所述收获EB和NPC（步骤2.6），并在室温下用4%多聚甲醛（PFA）溶液在1x PBS中固定它们30分钟。</li>
		<li>取出PFA并用1x PBS洗涤样品5分钟。</li>
		<li>将样品置于1x PBS，10%-，20%-和30%-蔗糖溶液的连续稀释液中，在25\u201228°C下，样品在孵育30分钟后转移到下一个溶液中。 注意:样品可以在继续包埋步骤之前，在4°C下储存在30%蔗糖溶液中。</li>
		<li>在将样品（不堆叠）置于冷冻模具的中心之前，用蔗糖溶液润湿移液器吸头，并移出多余的液体。 注:滤纸也可用于去除多余的溶液。用蔗糖溶液润湿移液器吸头对于防止EB和NPC粘附在吸头壁上非常重要。</li>
		<li>小心地将最佳切割温度（OCT）溶液添加到模具中，而不重复使用样品，并用移液器去除多余的气泡。</li>
		<li>将装有样品的模具以低速置于实验室混合器上，以轻度搅拌样品15分钟。这有助于将EB和NPC沉降到底部，如果它们重悬在OCT解决方案中。</li>
		<li>通过将模具置于液氮或干冰上，快速冷冻样品。 注意:样品可以储存在-70°C的冰箱中，然后再继续下一步。</li>
	</ol></li>
	<li>冷冻切除 
	<ol>
<li>将低温恒温器设置为冷却至-20至-18°C，然后将样品转移到仪器上。</li>
		<li>将冷冻的OCT块从模具中分离出来，并将其固定在支架上，并在支架表面上放置一点OCT溶液。</li>
		<li>对齐OCT块，使EB和NPC最接近刀片，以确保样品在切片过程中不会丢失。</li>
		<li>小心地切开10μm的块，并密切关注含有样品的切片。</li>
		<li>快速将含有样品的OCT切片置于组织包埋载玻片上，并允许OCT切片在室温下风干1小时。 注:样品可储存在-70°C供以后使用。</li>
	</ol></li>
	<li>免疫荧光 
	<ol>
<li>在室温下将含有神经元的OCT切片或培养室在1x PBS中阻断10%正常驴血清/ 0.1%Triton X-100中的1小时。</li>
		<li>将样品在稀释在5%正常驴血清/ 0.05%Triton X-100中的一抗中孵育在1x PBS中，在4°C下过夜。</li>
		<li>在1x PBS / 0.1%Triton X-100中洗涤样品三次，每次洗涤5分钟。</li>
		<li>将样品用稀释在5%正常驴血清/ 0.05%Triton X-100中的二抗在1x PBS中室温下孵育1小时。</li>
		<li>在1x PBS / 0.1%Triton X-100中洗涤样品三次，每次洗涤5分钟，然后将样品孵育在1μg/ mL DAPI中。</li>
		<li>用盖玻片和一些安装介质安装样品，并使其干燥。</li>
		<li>在荧光显微镜下观察样品。</li>
	</ol></li>
	<li>核糖核酸序列分析 
	<ol>
<li>收集不同阶段的细胞并进行RNA提取（见步骤3.2）。</li>
		<li>准备cDNA文库，根据Wang等人8中描述的方案进行深度测序和数据分析。</li>
		<li>使用 R 包 clusterProfiler 执行基因本体 （GO） 分析。</li>
	</ol></li>
	<li>流式细胞术 
	<ol>
<li>按照步骤1.6-1.7收集ESC，并将细胞重悬于培养基中。按照步骤2.4收集EB和NPC，并将细胞重悬于培养基中。</li>
		<li>使用40μm尼龙细胞过滤器过滤细胞悬浮液到新的15mL锥形管中。</li>
		<li>使用流式细胞仪（由机构的核心设施执行）测量样品的GFP信号。</li>
	</ol></li>
</ol>

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作者声明没有相互竞争的经济利益。

小鼠胚胎干细胞的神经分化方法已经建立了几十年，研究人员继续修改以前的方案或为各种目的创建新的方案7，10，11。我们利用一系列检测方法，综合分析了mESCs对神经元分化阶段的效率和进展，可用于分析小鼠或人类ESC的其他谱系分化。此外，我们的方法已被证明是评估特定基因或途径对体外神经元分化的影响的有用工具8。
通过我们的方法，用视黄酸（RA）处理的多能未承诺ESCs以高效率承诺神经谱系，并进一步诱导产生神经元7。为了提高ES细胞向神经细胞的成功分化并降低异质性，重要的是将ES细胞保持在未分化状态12。非增殖MEFs，用γ辐照或丝裂霉素C处理，功能维持ESC的多能性并为它们的生长提供支架13，14。为了获得一致的结果，我们使用在γ辐照MEF上培养的mESCs开始每个分化实验。经过几次传代后，γ辐照的MEF死亡，培养物最终成为mESC细胞的均质。或者，可以将mESC预镀在明胶上约45分钟，然后γ辐照的MEF接种，以便在下一次传代中更好地去除它们。白血病抑制因子（LIF）长期以来一直用于通过激活JAK / STAT途径15，16，17来维持培养的小鼠ES细胞的多能状态。最近，发现PD0325901（PD，一种MEK抑制剂）和CHIR99021（CH，一种GSK抑制剂）可提供ES细胞3，18的额外多能性维持。在我们的方案中，我们用这些抑制剂和LIF培养mESC，以保持mESC的高多能性。
实现成功差异化的另一个关键因素是EB的质量。我们通过悬挂滴法进行E14细胞的分化，其他研究者已经应用了5，19，20。使用这种方法，允许单个ES细胞悬浮在分化培养基液滴中2天，在那里它们自发聚集并形成EB。与在介质中悬浮分离的mESC的方法相比，所得到的EB在形态方面通常更明确（图2B），这导致EB尺寸在我们的经验中更宽（数据未显示）。为了防止EB附着在板上，重要的是从第3天开始进行低速旋转，一直持续到分化过程。EB通过用RA治疗来诱导它们分化成NPC。EB第4天RA治疗产生的NPC对于神经细胞（如少突胶质细胞和星形胶质细胞21）通常是异质性的。使用RT-qPCR或免疫荧光，可以探测鼻咽癌群体的神经元和其他神经谱系标志物，例如星形胶质细胞的Gfap和少突胶质细胞的Olig2。为了进一步诱导NPC分化为神经元，NPC在最佳神经元培养基中培养，其中最重要的成分是N2和B27补充剂。N2补充剂的主要功能是帮助NPC致力于神经元谱系，而B27补充剂的功能是维持神经元的寿命。
样本可以在分化期（例如，ESC阶段，EB第2天，EB第4天，NPC第6天，NPC第8天，神经元第10天和神经元第12天）收集，通过对多能性和外胚层标志物进行RT-qPCR来跟踪分化过程。比较不同细胞阶段之间的多能性标记，如Oct4、Nanog、Sox2、Klf4和Myc，将验证mESC的多能性（图3B，F）。 为了研究神经分化的效率，中胚层的标志物如Hand1，Snai1和Tbxt; 和内胚层，如Eomes和Gata4也可以探测（数据未显示）。进一步的验证可以通过免疫荧光（IF）探测鼻咽癌或神经元标志物进行（图3A）。然而，这些方法不是定量的，并且偏向于选定的标记。为了克服这些限制，我们结合了流式细胞术和RNA-seq分析（图3C\u2012F）。流式细胞术实验中使用的细胞系是Sox1-GFP E14细胞系，该细胞系专门用于评估鼻咽癌分化程序的质量。Sox1是神经外胚层发育过程中最早的特定神经元标志物之一22，因此使其成为鼻咽癌谱系的极好标志物。Sox1可以探测使用RT-qPCR或蛋白质印迹来评估鼻咽癌群体。这些分析特别有利于研究由基因操作或化学处理引起的分化缺陷。
重要的是要注意，这里介绍的协议有一些限制。首先，我们仅介绍一种野生型mESC细胞系的全面分析。来自小鼠或人类的其他ESC系可能需要在方案中进行更改和进一步优化，以确保成功和有效的神经元分化。其次，我们提出了一种体外神经元分化方法，这种方法自然有其自身的局限性。如前所述，EB接受超生理水平的RA治疗，以驱动它们走向鼻咽癌谱系。然后将产生的NPC放置在神经元最佳培养基中，以模仿生理条件并鼓励神经元谱系承诺，生长和长寿。在这里，N2和B27补充剂用于培养神经元，但也可以使用其他补充剂，例如用于类似目的的NS21 23，这可能会改变神经元分化的成功和效率。这些条件在细胞培养测定中合成重建，这可能不完全代表生理条件。EB，NPC和神经元的质量在很大程度上取决于起始mESC。已经传代次数过多并在培养物中保存超过1周的mESCs通常开始失去多能性，并且可能无法成功进行分化。因此，将mESC保持在最佳状态是确保它们能够有效分化为EB，NPC和神经元的关键。其他神经元培养方法，如3D模型也被提出来更好地模仿生理条件24，25，26，有时以牺牲通量和可行性为代价27，28。我们相信我们的实验方案可用于表征这些3D培养模型。

自从它们第一次从发育中的小鼠囊胚1、2的内细胞质量中衍生出来以来，小鼠胚胎干细胞（mESC）已被用作研究干细胞自我更新和分化的强大工具3。此外，研究mESC分化导致对分子机制的深刻理解，这些机制可以提高基于干细胞的治疗治疗神经退行性疾病等疾病的效率和安全性4。与动物模型相比，这种体外系统具有许多优点，包括实践和评估的简单性，与动物相比，维持细胞系的成本较低，以及遗传操作的相对容易性。然而，分化细胞类型的效率和质量往往受到不同的mESCs系以及分化方法5，6的影响。此外，评估分化效率的传统检测依赖于对所选标记基因的定性检查，这些标记基因缺乏稳健性，因此无法掌握基因表达的全局变化。
在这里，我们的目标是使用一系列测定来系统地评估神经元分化。使用对所选标记物和RNA-seq的传统体外分析，我们建立了一个平台来测量分化效率以及在此过程中的转录组学变化。基于先前建立的方案7，我们通过悬挂滴技术产生类胚体（EB），然后使用超生理量的视黄酸（RA）诱导产生神经祖细胞（NPC），随后用神经诱导培养基分化为神经元。为了检查分化的效率，除了传统的RT-qPCR和免疫荧光（IF）测定外，我们还进行了RNA-seq和流式细胞术。这些分析提供了对阶段特异性分化进展的全面测量。

<table><tbody><tr><td>0.05% Trypsin + 0.53mM EDTA 1X</td><td>Corning</td><td>25-052-CV</td><td></td></tr><tr><td>0.1% Gelatin</td><td>Sigma</td><td>G1890-100G</td><td>Prepared in de-ionized water</td></tr><tr><td>16% Paraformaldehyde</td><td>Thermo Scientific</td><td>28908</td><td>Diluted in 1X PBS</td></tr><tr><td>40-&amp;mu;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Albumax</td><td>Thermo Fisher Scientific</td><td>11020021</td><td></td></tr><tr><td>AlexaFluor 488 goat anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A11001</td><td>Antibody was diluted at 1:500 for IF</td></tr><tr><td>Alkaline Phosphatase Staining Kit II</td><td>Stemgent</td><td>00-0055</td><td></td></tr><tr><td>AzuraQuant Green Fast qPCR Mix LoRox</td><td>Azura Genomics</td><td>AZ-2105</td><td></td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>BD FACSCanto</td><td>BD</td><td>657338</td><td></td></tr><tr><td>bFGF</td><td>Sigma</td><td>11123149001</td><td></td></tr><tr><td>BioAnalyzer High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Chir99021</td><td>Cayman Chemicals</td><td>13122</td><td></td></tr><tr><td>Chloroform</td><td>C298-500</td><td>Fisher Chemical</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>R37606</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CM</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11320033</td><td></td></tr><tr><td>EB buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>Ethanol</td><td>111000200</td><td>Pharmco</td><td>Diluted in de-ionized water</td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S10250</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>HiSeq 2500 Sequencing System</td><td>Illumina</td><td>SY-401-2501</td><td></td></tr><tr><td>Isopropanol</td><td>BDH1133-4LG</td><td>BDH VWR Analytical</td><td>Diluted in de-ionized water</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030024</td><td></td></tr><tr><td>LIF</td><td>N/A</td><td>N/A</td><td>Collected from MEF supernatant</td></tr><tr><td>m18srRNA primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-GCAATTATTCCCCATGAACG-3&#039;&lt;br/&gt; 5&#039;-GGCCTCACTAAACCATCCAA-3&#039;</td></tr><tr><td>MEM Non-essential amino acids</td><td>Corning</td><td>25-025-Cl</td><td></td></tr><tr><td>mNanog primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGGCTTTGGAGACAGTGAGGTG-3&#039;&lt;br/&gt; 5&#039;-TGGGTAAGGGTGTTCAAGCACT-3&#039;</td></tr><tr><td>mNes primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGTGCCCAGTTCTAGTGGTGTCC-3&#039;&lt;br/&gt; 5&#039;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#039;</td></tr><tr><td>mNeuroD1 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-CGAGTCATGAGTGCCCAGCTTA-3&#039;&lt;br/&gt; 5&#039;-CCGGGAATAGTGAAACTGACGTG-3&#039;</td></tr><tr><td>mOct4 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGATCACTCACATCGCCAATCA-3&#039;&lt;br/&gt; 5&#039;-CGCCGGTTACAGAACCATACTC-3&#039;</td></tr><tr><td>mPax6 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-CTTGGGAAATCCGAGACAGA-3&#039;&lt;br/&gt; 5&#039;-CTAGCCAGGTTGCGAAGAAC-3&#039;</td></tr><tr><td>N2 supplement</td><td>Thermo Fisher Scientific</td><td>17502048</td><td></td></tr><tr><td>Nestin primary antibody</td><td>Millipore</td><td>MAB5326</td><td>Antibody was diluted at 1:200 for IF</td></tr><tr><td>Neural basal</td><td>Thermo Fisher Scientific</td><td>21103049</td><td></td></tr><tr><td>Neurofilament primary antibody</td><td>DSHB</td><td>2H3</td><td></td></tr><tr><td>NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td><td>BioO Scientific</td><td>NOVA-5138-07</td><td></td></tr><tr><td>PD0325901</td><td>Cayman Chemicals</td><td>13034</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>N/A</td><td>N/A</td><td>Prepared in de-ionized water</td></tr><tr><td>- Potassium chloride</td><td>P217-500G</td><td>VWR</td><td></td></tr><tr><td>- Potassium phosphate monobasic anhydrous</td><td>0781-500G</td><td>VWR</td><td></td></tr><tr><td>- Sodium chloride</td><td>BP358-10</td><td>Fisher Bioreagents</td><td></td></tr><tr><td>- Sodium phosphate, dibasic, heptahydrate</td><td>SX0715-1</td><td>Milipore</td><td></td></tr><tr><td>Random hexamer primer</td><td>Thermo Scientific</td><td>SO142</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>Prepared in DMSO</td></tr><tr><td>Sodium pyruvate</td><td>Corning</td><td>25-000-Cl</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>84097</td><td>Diluted in 1X PBS</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Invitrogen</td><td>18064022</td><td></td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>TriPure Isolation Reagent</td><td>Sigma-Aldrich</td><td>11667165001</td><td></td></tr><tr><td>TruSeq Rapid</td><td>Illumina</td><td>20020616</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Fisher BioReagents</td><td>BP176-100</td><td></td></tr></tbody></table>

differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells

我们描述了将小鼠胚胎干细胞培养和分化为神经元谱系的分步程序，然后进行一系列测定以表征分化细胞。利用E14小鼠胚胎干细胞通过悬挂滴法形成胚体，然后通过视黄酸诱导分化为神经祖细胞，最后分化成神经元。定量逆转录聚合酶链反应（RT-qPCR）和免疫荧光实验表明，神经祖细胞和神经元分别在分化后第8天和第12天表现出相应的标志物（神经祖的巢巢蛋白和神经元的神经丝）。在表达Sox1启动子驱动的GFP报告基因的 E14 系上进行流式细胞术实验表明，第8天约60%的细胞为GFP阳性，表明该阶段神经祖细胞成功分化。最后，使用RNA-seq分析来分析全局转录组学变化。这些方法可用于分析特定基因和途径在神经元分化过程中调节细胞身份转变的参与。

作为我们方法的代表，我们在E14细胞上进行了EB，NPC和神经元分化实验。E14细胞在γ辐照的MEF上培养（图1A），直到γ辐照的MEF群体被稀释。我们通过对Nanog和Oct4标记物进行碱性磷酸酶（AP）染色（图1B）和后来的RT-qPCR（见下文）证实了E14细胞的多能性。然后使用图2A中概述的方案诱导γ照射的不含MEF的E14细胞进行分化。简而言之，将含有500个细胞的20μL分化培养基液滴接种在培养板的盖子上（有关详细信息，请参见方案部分2）。然后将形成的EB收集并置于新鲜分化培养基中的悬浮液中。从分化的第4天到第8天，将5μM RA加入培养板以诱导NPC.分化的EB显示出圆形，并且在分化过程中其大小继续增加（图2B）。在第8天，收获NPC并进行胰蛋白酶消化，然后将所得的单细胞悬浮液接种在DEM / F12培养基中的组织培养室中，加入N2补充剂，然后加入B27补充剂。到第10天，分化成神经元的NPC似乎具有细长的细胞形状（图2B）。
为了进一步评估我们的分化实验，我们在第8天对E14 NPC进行了免疫荧光（IF）实验，在第12天对E14神经元进行了免疫荧光（IF）实验。我们观察到NPC中巢蛋白的阳性染色和神经元的神经丝（NF）信号（图3A）。或者，RT-qPCR和RNA-seq证实了NPC标记基因的诱导和NPC中多能基因的丧失（图3B，D，F）。作为测试ESC分化成功的定量方法，我们分化了表达 Sox1 启动子驱动的GFP报告基因9的小鼠ESC系，然后对ESC和NPC进行流式细胞术分析。我们发现，在鼻咽癌阶段，总细胞的58.7%是GFP阳性，而在ESC阶段，GFP信号为0.0%（图3C）。为了分析分化过程中的转录组学变化，进行了E14 ESCs，EB第3天和NPC第8天的RNA-seq实验，并揭示了与相应阶段相关的基因簇（图3D）。根据RNA-seq热图中的基因的表达水平对基因进行排序，以鉴定分化过程中不同阶段的差异表达基因。对四个基因簇的基因本体（GO）分析表明，这些簇对应于不同的细胞功能或途径，表明mESC神经元分化的三个细胞阶段各自具有一组在各自阶段高度表达的基因，而不是其他基因（图3E）。例如，与其他阶段相比，簇3中的基因在E14 NPC中高度表达，并且对应于与神经元发育相关的途径。簇1，2和4不包含与任何生殖层谱系规格相关的高表达基因，但它们与细胞生长和增殖有关。因此，RNA-seq和伴随的GO分析表明，E14细胞在分化的第8天已经分化成神经元谱系。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61446/61446fig1.jpg"> 图1:培养物中的E14 ESC。 （A）光学显微镜图像显示E14细胞（黑色箭头）生长在γ辐照MEF顶部的菌落中。E14菌落继续增殖，如第1天和第3天培养物之间的菌落大小差异所示。（B） 通过碱性磷酸酶 （AP） 染色确认 E14 ESC 的多能性。紫色箭头表示 AP 染色阳性的 mESC。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig1large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61446/61446fig2.jpg"> 图2:E14分化为EB，NPC和神经元。 （A） 示意图总结了将 E14 细胞分化为 EB、NPC 和神经元的主要步骤。（B）E14 ESCs在没有LIF的培养基中培养，2i在悬浮板中形成单独的EB球体，在第2天可见，在随后的几天内继续生长和膨胀。在分化的第4天加入RA以诱导分化为NPC。诱导4天后，这些NPC被接种以分化成神经元，这些神经元显示在底部面板中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61446/61446fig3.jpg"> 图3:分化细胞的表征。（A） 顶部面板中的免疫荧光图像显示，在第8天探测了含有鼻咽癌的EB，以检测巢蛋白（绿色）和细胞核（DAPI，蓝色）。下图显示了第12天探测神经丝的神经元的免疫荧光图像（绿色）。合并图像中的红框将缩放 3 倍以获得更好的视图。（B）RT-qPCR分析显示E14 ESC和NPC的多能性标记（Nanog和Oct4）和N ±PC标记（Pax6，NeuroD1和Nes）。 第8天的ESC和NPC都通过流式细胞术定量为阳性GFP荧光信号。（D）热图显示了在ESC，EB和NPC阶段表达的基因的确定FPKM的z评分。鉴定出四个不同的基因簇，表示在ESC，EB或NPC阶段差异表达的基因组。（E） GO分析是使用R包clusterProfiler对RNA-seq中鉴定的四个簇进行的。（F） 该图显示了其他三个多能性标记的FPKM值，Sox2，Klf4和Myc，以及神经标记，NeuN，Map2和Tubb3在ESC，EB第3天和NPC第8天阶段的E14细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

我们描述了使用悬挂滴法将小鼠胚胎干细胞体外分化为神经元细胞的过程。此外，我们通过RT-qPCR，免疫荧光，RNA-seq和流式细胞术进行全面的表型分析。

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

differentiation-characterization-neural-progenitors-neurons-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

4.9K 次观看.  Penn State Hershey Cancer Institute. 我们描述了使用悬挂滴法将小鼠胚胎干细胞体外分化为神经元细胞的过程。此外，我们通过RT-qPCR，免疫荧光，RNA-seq和流式细胞术进行全面的表型分析。

视频: Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

科研

发育生物学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

神经科学

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

生物学

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Source: Hanafiah, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61446">Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells.</a> J. Vis. Exp. (2020).
The video demonstrates the differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) by the hanging drop method. Upon suspending the mESCs in hanging drops and incubating, the cells aggregate to form EBs, which are further differentiated into neuronal progenitor cells (NPCs) using a differentiation medium supplemented with retinoic acid (RA).

通过 Hang Drop 方法从小鼠胚胎干细胞生成神经祖细胞

1. mESC culture

	Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

将人类胚胎干细胞分化为神经祖细胞

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

Advanced Neuronal Models

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

一种从小鼠胚胎干细胞生成神经祖细胞的技术

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.