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<s...

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 border="0" cellpadding="0" cellspacing="0" style="width:805px;" width="804">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.05% Trypsin + 0.53mM EDTA 1X</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-052-CV</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.1% Gelatin</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">G1890-100G</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">16% Paraformaldehyde</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">28908</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">40-&#956;m cell strainer</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:127px;">352340</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Albumax</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11020021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">AlexaFluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">A11001</td>
			<td>Antibody was diluted at 1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:148px;">Stemgent</td>
			<td style="width:127px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">AzuraQuant Green Fast qPCR Mix LoRox</td>
			<td style="width:148px;">Azura Genomics</td>
			<td style="width:127px;">AZ-2105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">B27 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BD FACSCanto</td>
			<td style="width:148px;">BD</td>
			<td style="width:127px;">657338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">bFGF</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">11123149001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BioAnalyzer High Sensitivity DNA Kit</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:127px;">5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chir99021</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chloroform</td>
			<td style="width:148px;">C298-500</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DAPI</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">R37606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DMEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">DMEM/F12 medium</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">EB buffer</td>
			<td style="width:148px;">Qiagen</td>
			<td style="width:127px;">19086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethanol</td>
			<td style="width:148px;">111000200</td>
			<td style="width:127px;">Pharmco</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Fetal bovine serum</td>
			<td style="width:148px;">Atlanta Biologicals</td>
			<td style="width:127px;">S10250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">HiSeq 2500 Sequencing System</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">SY-401-2501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Isopropanol</td>
			<td style="width:148px;">BDH1133-4LG</td>
			<td style="width:127px;">BDH VWR Analytical</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">L-glutamine</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">25030024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">LIF</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Collected from MEF supernatant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">m18srRNA primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-GCAATTATTCCCCATGAACG-3&#39; 
			5&#39;-GGCCTCACTAAACCATCCAA-3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MEM Non-essential amino acids</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mNanog primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGGCTTTGGAGACAGTGAGGTG-3&#39; 
			5&#39;-TGGGTAAGGGTGTTCAAGCACT-3&#39;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">mNes primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGTGCCCAGTTCTAGTGGTGTCC-3&#39; 
			5&#39;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mNeuroD1 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CGAGTCATGAGTGCCCAGCTTA-3&#39; 
			5&#39;-CCGGGAATAGTGAAACTGACGTG-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mOct4 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGATCACTCACATCGCCAATCA-3&#39; 
			5&#39;-CGCCGGTTACAGAACCATACTC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mPax6 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CTTGGGAAATCCGAGACAGA-3&#39; 
			5&#39;-CTAGCCAGGTTGCGAAGAAC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">N2 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nestin primary antibody</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:127px;">MAB5326</td>
			<td>Antibody was diluted at 1:200 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Neural basal</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">21103049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Neurofilament primary antibody</td>
			<td style="width:148px;">DSHB</td>
			<td style="width:127px;">2H3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td>
			<td style="width:148px;">BioO Scientific</td>
			<td style="width:127px;">NOVA-5138-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">PD0325901</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Penicillin/streptomycin</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">- Potassium chloride</td>
			<td style="width:148px;">P217-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Potassium phosphate monobasic anhydrous</td>
			<td style="width:148px;">0781-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium chloride</td>
			<td style="width:148px;">BP358-10</td>
			<td style="width:127px;">Fisher Bioreagents</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium phosphate, dibasic, heptahydrate</td>
			<td style="width:148px;">SX0715-1</td>
			<td style="width:127px;">Milipore</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Random hexamer primer</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">SO142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Retinoic acid</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">R2625</td>
			<td>Prepared in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium pyruvate</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-000-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sucrose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">84097</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">SuperScript III Reverse Transcriptase</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">18064022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:148px;">Sakura</td>
			<td style="width:127px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TriPure Isolation Reagent</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:127px;">11667165001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TruSeq Rapid</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">20020616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">&#946;-mercaptoethanol</td>
			<td style="width:148px;">Fisher BioReagents</td>
			<td style="width:127px;">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?...

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

Research

JoVE Journal

Developmental Biology

4.7K 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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...