这项工作得到了中国国家自然科学基金委员会（第31501099号）和中国湖北省教育厅中青年（第31501099号）的支持。Q20191104）。我们感谢武汉科技大学邓文生教授提供小鼠胚胎干细胞系A2lox。

1. 小鼠胚胎干细胞培养
<ol><li>准备 0.1% 明胶涂层细胞培养皿或盘子。<ol>
<li>加入2 mL的灭菌0.1%明胶（0.1%w/v在水中）到60毫米细胞培养皿。轻轻摇动，确保细胞培养皿均匀涂层。</li>
		<li>将菜肴放入 37 °C 下的 5% CO2 孵化器中，并允许涂层 1 小时。</li>
		<li>在播种细胞之前取出0.1%的明胶溶液。 注意:去除明胶后，无需干燥或清洗涂层的盘子。</li>
	</ol>
</li>
	<li>小鼠胚胎干细胞（A2lox和129）培养<ol>
<li>在 0.1% 明胶中孵化 mESC （A2lox 和 129） 细胞，分别在 5% CO2 孵化器中以 37 °C 在 mESC 生长介质中涂上 60 mm 细胞培养皿。mESC 增长介质包括 85% 淘汰 DMEM/F12、15% 淘汰血清置换 （KSR）、0.1 m β甲醇 （2ME）、2 mM 谷胱甘肽、 1%非必需氨基酸（NEAA）、1%青霉素/链霉素（P/S）、1000 U/mL白血病抑制因子（LIF）、10 nM CHIR-99021（GSK-3抑制剂）和0.33 nM PD0325901（MEK抑制剂）。 警告:β甲醇易燃，具有吸入毒性。远离火源，戴上口罩，避免在使用时吸入。</li>
		<li>每天更改 mESC 增长介质，以更好地促进 A2lox 和 129 的增长。</li>
		<li>当细胞达到80%的汇合时，取出介质，在菜中加入1 mL的0.1%的试金石。轻轻摇动30s，以确保所有细胞上均覆盖试金石。</li>
		<li>离开细胞约1分钟 ，尝试蛋白化，然后使用1 mL移液器去除试金石。</li>
		<li>在菜中加入 2 mL 的 mESC 生长介质，上上下下几次移液器，使单个细胞悬架。</li>
		<li>使用血细胞计尽可能准确地计算悬架中细胞的密度。</li>
		<li>将细胞分成7组，使用 表1中显示的不同方案诱导分化。</li>
	</ol>
</li>
</ol>2. 从中小企业到NPC的差异化（第一阶段）
<ol><li>准备 0.1% 明胶涂层细胞培养板或盖片。<ol>
<li>使用前，准备 0.1% 明胶涂层 6 井板或盖片，如第 1.1 步。</li>
	</ol></li>
	<li>使用协议 1 （表 1）进行第一阶段分化<ol>
<li>种子约2 x 104 mESCs在2 mL的基底分化介质I每口井在0.1%明胶涂层6井板。检查显微镜下的细胞密度。</li>
		<li>在 5% CO2 孵化器中以 37 °C 孵育细胞 6 小时，以便附件。</li>
		<li>附着后，从孵化器中取出细胞，用2 mL的PBS清洗细胞两次。</li>
		<li>在每个井中加入2 mL的基底分化介质I（表1），并将细胞放回孵化器。</li>
		<li>离开细胞进行8天的分化。每2天更换一次基底分化介质I。</li>
	</ol></li>
	<li>使用协议 2 （表 1） 进行第一阶段分化<ol>
<li>在 10 mL 的基底分化介质 I 中加入 1.5 x 106 mESCs 到非粘性细菌盘中，以便在 5% CO2 孵化器中以 37 °C 形成胚胎体。</li>
		<li>2天后，将细胞聚集物转移到15mL离心管中，让它们通过重力沉降。</li>
		<li>去除超纳坦，加入10mL的新鲜基底分化介质I，以重新悬浮胚胎体。将它们重新种植到一个新的非粘性细菌盘中，并允许分化2天。</li>
		<li>检查显微镜下胚胎体的形成（图2A）。</li>
		<li>收集步骤 2.3.2-2.3.3 中描述的胚胎体。种子约50胚胎体在2mL的基底分化介质I每口井到0.1%明胶涂层6井板。</li>
		<li>准备 1 mm 全跨 RA 库存（在 DMSO 中），并在分包装后将光线存放在 -80 °C 冰柜中。 注意:RA是不稳定的，在准备RA库存时，应注意使其远离光线和减少空气接触。</li>
		<li>对于 RA 感应，在每个油井中加入 2 μL 的 RA 库存，最终浓度为 1 μM。</li>
		<li>将板放入 37 °C 的 5% CO2 孵化器中，再分化 4 天。</li>
		<li>每2天更换2 mL的基底分化介质I（带1μM RA）。</li>
	</ol></li>
	<li>使用协议 3 （表 1） 分化第一阶段<ol>
<li>将 1.5 x 106 mESCs 种植到 10 mL 的基底分化介质 I 中，在 5% CO 2 孵化器中以 37 °C 的胚胎体形成2 天。</li>
		<li>检查显微镜下胚胎体的形成（图2B）。</li>
		<li>将细胞聚集物转移到15 mL离心管中，让它们通过重力稳定下来。</li>
		<li>小心地取出超纳坦，加入10 mL的新鲜基底分化介质I来重新悬浮它们。</li>
		<li>种子约50胚胎体到2 mL的基底分化介质I每口井到0.1%明胶涂层6井板。</li>
		<li>对于 RA 感应，在每个油井中加入 2 μL 的 RA 库存，最终浓度为 1 μM。</li>
		<li>将板放入 37 °C 下的 5% CO2 孵化器中，再分化 6 天。每 2 天更换一次整个基底分化介质 I（带 1 μM RA）。</li>
	</ol></li>
	<li>使用协议 4 （表 1） 进行第一阶段分化<ol>
<li>种子约 2 x 104 mESCs 在 2 mL 的基底分化介质 I 每口井上 0.1% 明胶涂层 6 井板。将板放入 37 °C 下的 5% CO2 孵化器中，以便附件 6 小时。</li>
		<li>附件后，用2 mL的PBS清洗细胞两次。将 2 mL 的基底分化介质 I 添加到每个井中，并在 37 °C 的 5% CO2 孵化器中分化 4 天。</li>
		<li>对于 RA 感应，在每个井中添加 2 μL 的全跨 RA 库存（工作浓度为 1 μM），以诱导分化再持续 4 天。</li>
		<li>在整个过程中，每2天更换一次整个媒体。</li>
	</ol></li>
	<li>使用协议 5 （表 1） 进行第一阶段分化<ol>
<li>种子约 2 x 104 mESCs 在 2 mL 的基底分化介质 I 每口井上 0.1% 明胶涂层 6 井板。将板放入 37 °C 下的 5% CO2 孵化器中，以便附件 6 小时。</li>
		<li>用 2 mL 的 PBS 清洗细胞两次。将 2 mL 的基底分化介质 I 添加到每个井中，并在 37 °C 的 5% CO 2 孵化器中进行2 天的分化。</li>
		<li>对于后续的 RA 感应，在每口油井中加入 2 μL 的 RA 库存，使最终浓度为 1 μM。 将板放入 37 °C 下的 5% CO2 孵化器中，以诱导分化再持续 6 天。</li>
		<li>在整个过程中，每2天更换一次整个媒体。</li>
	</ol></li>
	<li>使用协议 6 （表 1） 进行第一阶段分化<ol>
<li>在N2B27介质II（表1）的10mL中将1.5 x 106 mESCs植物成非粘性细菌盘，以允许胚胎体的形成。</li>
		<li>第2天，收集步骤2.3.2-2.3.3中描述的细胞聚合物，并使用10 mL的新鲜N2B27介质II重新悬生胚胎体。</li>
		<li>将它们重新种植到一个新的非粘性细菌盘中，并在37°C的5%CO2 孵化器中再分化2天。 检查显微镜下胚胎体的形成。</li>
		<li>第4 天，收集胚胎体。种子约50胚胎体每井0.1%明胶涂层6井板与2 mL的N2B27中等II。</li>
		<li>将 2 μL 的全跨 RA 库存添加到每个井中，并诱导分化再持续 4 天。每两天更换一次整个介质（N2B27 介质 II 与 1 μM RA）。</li>
	</ol></li>
	<li>使用协议 7 （表 1） 进行第一阶段分化<ol>
<li>种子约 2 x 104 mESCs 在 2 mL 的基底分化介质 I 每口井上 0.1% 明胶涂层 6 井板。将板放入 37 °C 下的 5% CO2 孵化器中，以便附件 6 小时。</li>
		<li>用PBS清洗细胞两次。然后，在每个油井中添加 2 mL 的 N2B27 介质 II，并在 5% CO2 孵化器中在 37 °C 下进行 8 天的分化。</li>
		<li>每2天更换一次整个N2B27介质II。</li>
	</ol></li>
</ol>3. 细胞形态观察
<ol><li>每天在倒相对比光显微镜下检查上述7组的分化状态。</li>
	<li>随机选择至少 12 个字段并拍照以记录 D8 上每个组的形态变化。</li>
</ol>4. 免疫荧光染色
<ol><li>样品制备:0.1%明胶涂层盖片上的种子 mESC，并允许使用第 2 步中提及的协议进行 8 天的分化。</li>
	<li>冲洗:第8天 ，从孵化器中取出样品，并按吸入去除分化介质。用 1 mL 的 PBS 轻轻冲洗细胞一次 5 分钟。</li>
	<li>固定:在每个样品中添加4%的准甲醛的1mL，并在室温下固定细胞20分钟（RT）。</li>
	<li>冲洗:固定后，用1 mL的PBS轻轻冲洗细胞3次，每次5分钟。</li>
	<li>渗透:在 PBS 中添加 1 mL 的 0.2% 三元 X-100，并在 RT 中保留 8 分钟。</li>
	<li>冲洗:渗透后，用1mL的PBS轻轻冲洗细胞3次，每次5分钟。</li>
	<li>阻止:在PBS中添加1 mL的10%山羊血清到每个样本中，并在RT孵育1小时，以阻止任何非特定的相互作用。</li>
	<li>与原发性抗体的孵化<ol>
<li>使用PBS中5%的山羊血清以1:100的比例稀释抗内斯汀抗体。</li>
		<li>将500μL的稀释抗体涂抹到不同的样品上，并在4°C下孵育过夜。</li>
	</ol>
</li>
	<li>冲洗:去除抗体，用1mL的PBS轻轻冲洗样品，每次8分钟。</li>
	<li>二次抗体孵育<ol>
<li>在PBS中使用5%的山羊血清，以1:500的比例稀释Alexa Fluor 488标签的山羊抗鼠IgG。</li>
		<li>将500μL的稀释抗体涂抹在不同的样品上，并在RT中在黑暗中孵育2小时。 注:应用荧光二次抗体后，在黑暗中执行所有后续步骤，以防止荧光淬火。</li>
	</ol>
</li>
	<li>冲洗:去除二次抗体，用1 mL的PBS轻轻冲洗样品，每次8分钟。</li>
	<li>核污渍和安装<ol>
<li>将一滴 DAPI 安装介质放置在干净的微滑板上。</li>
		<li>小心地从板中取出样品，并将样品放在 DAPI 安装介质顶部，细胞面朝下。在黑暗中离开 5 分钟在 Rt 。</li>
		<li>用吸和纸去除多余的 DAPI 安装介质。</li>
	</ol>
</li>
	<li>荧光显微镜观察<ol>
<li>将标本置于荧光显微镜下，使用适当的过滤器检测 DAPI 和 Alexa Fluor 488 的信号。</li>
		<li>为每个样本均匀随机地选择 10-15 个不同的视场，并使用 CCD 摄像机记录图像。</li>
	</ol>
</li>
</ol>5. 从 NPC 到神经元的差异（第二阶段）
<ol><li>使用协议 3（8 天，表 1）在 I 阶段分化下准备 mESC 衍生工具，详见步骤 2.4，具有最高的分化效率（见图 3）。 注:第一阶段分化后，应采用上述细胞形态观察和免疫荧光检测进行质量控制，以确保NPC健康高产。</li>
	<li>种子约 5 x 105 mESC 衍生物在 2 mL 的基底分化介质 I 每口井上 0.1% 明胶涂层 6 井板。随机将 mESC 衍生工具分为 3 组，分别是第二阶段协议 1、协议 2 和协议 3。</li>
	<li>将板放入 37 °C 下的 5% CO2 孵化器中，以便附件 6 小时。用2 mL的PBS清洗两次。</li>
	<li>在上述组的每口井中分别添加2 mL的基底分化介质I、N2B27介质I和N2B27介质II（表2）。</li>
	<li>将板放入孵化器中，并允许再区分 10 天。每2天更换一次相应的介质。</li>
	<li>检查分化状态并记录第 3 步中提到的形态变化。</li>
	<li>在第18天，评估神经元的生成（β-Tubulin III阳性），并确定第4步中使用的3个协议的分化效率。</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+upregulation+of+miR-219+promotes+the+differentiation+of+embryonic+stem+cells+into+neural+cells.">Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells.</a> Cell Death Disease. 8, 2953 (2017).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+regulates+germ+cell+differentiation+in+mouse+embryonic+stem+cells+through+a+Smad-dependent+pathway.">Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.</a> Biochemical and Biophysical Research Communications. 418 (3), 571-577 (2012).</li><li>Nakayama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+neuronal+induction+of+the+P19+embryonic+carcinoma+cell+line.">A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.</a> Journal of Neuroscience Methods. 227, 100-106 (2014).</li><li>Bibel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature neuroscience. 7 (9), 1003-1009 (2004).</li><li>Coyle, D. E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

作者没有什么可透露的。

在本研究中，我们建立了一种简单而有效的神经元分化方法，具有成本低、材料易用等功能。在这种方法中，2天的胚胎体形成，然后是6天的RA诱导，可以有效地促进mESC在NPC中的分化（第一阶段协议3）。对于二期分化，N2B27介质II（II-协议3）最有效地诱导从NPC分化为神经元。为确保成功，应更多地注意几个关键步骤。
首先，胚胎体的健康状态是整个分化过程的关键。三维胚胎...

胚胎干细胞（ESCs）是多能的，可以分化成神经前体细胞（NPC），随后在某些条件下进入神经元1。基于ESC的神经生成提供了模仿神经生成的最佳平台，从而作为发展生物学研究的有用工具，并可能有助于再生医学2，3。在过去的几十年里，许多诱导胚胎神经生成的策略被报道，如转基因方法4，使用小分子5，使用3D矩阵微环境6，和共生技术7。但是，这些协议大多条件有限或难以操作，因此不适合在大多数实验室中使用。
为了找到一种操作简单、成本低廉的方法，实现与 mESC 的有效神经分化，这里使用了组合筛查策略。如图1所述，胚胎神经生成的整个过程分为两个阶段。第一阶段是指从 mESC 到 NPC 的分化过程，第二阶段涉及从 NPC 到神经元的后续分化。基于操作方便、成本低、材料易用、分化效率高的原则，根据传统的单层培养系统或胚胎体形成系统8、9，选择了第一阶段的7个协议和第二阶段的3个协议。利用细胞形态观察和免疫荧光检测，评估了两个阶段协议的分化效率。通过结合每个阶段最有效的协议，我们建立了神经分化与 mESC 的优化方法。

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

小鼠胚胎干细胞（mESCs）的神经分化是阐明神经生成的关键机制并可能有助于再生医学的潜在工具。在这里，我们建立了一种高效和低成本的神经元分化方法，从mESC体外， 使用组合筛选的策略。根据此处定义的条件，2 天胚胎体形成 – 6 天视网膜酸诱导协议允许从 mESC 快速有效地分化为神经前体细胞 （NPC），如形成堆叠良好和中性状 A2lox 和 129 个内斯汀阳性衍生物所示。胚胎体的健康状态和使用网膜酸 （RA） 的时间点以及 RA 浓度在此过程中至关重要。在随后从NPC向神经元的分化中，N2B27中II（辅以神经基质介质）可以更好地支持神经元细胞的长期维持和成熟。该方法效率高、成本低、操作方便，是神经生物学和发育生物学研究的有力工具。

2 天胚胎体形成 + 6 天 RA 感应最能指导 MESC 分化到 NPC （第一阶段）。为了确定最能促进将 mESC 分化为 NPC（第一阶段）的最佳方案，在 A2lox 和 129 mESC （表 1）上测试了 7 个协议，并使用光显微镜监测了每个组的分化状态。如 图3A所示，在"2天胚胎体形成+6天RA诱导"治疗下，大多数A2lox和129个衍生物（第一阶段协议3）显示堆积良好和中性状形态，这表明NPC的形成。?...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

在这里，我们建立了一种低成本和易于操作的方法，将胚胎干细胞快速高效分化为神经元。该方法适用于实验室的推广，是神经学研究的有用工具。

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 ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.8K 次观看.  Wuhan Center for Disease Control and Prevention. 在这里，我们建立了一种低成本和易于操作的方法，将胚胎干细胞快速高效分化为神经元。该方法适用于实验室的推广，是神经学研究的有用工具。

视频: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化:一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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 ...

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

小鼠星形胶质细胞的分离和直接神经元重编程

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...