我们感谢 Nkx2.1 的发展:: mCherry: Lhx6: GFP 双重记者制线以及詹妮弗. 泰森、Maroof 和蒂姆. 彼得罗斯米为帮助开发这一协议而进行的早期工作。我们还感谢印章流式细胞仪的核心技术援助。这项工作得到了 NIH R01 MH066912 (SA) 和 F30 MH105045-02 (DT) 的支持。

注: 本协议中描述的双记者制线可根据要求提供 (sande@mail.med.upenn.edu sande@mail 大学)。
1. 媒体准备
注意: 在细胞培养使用前, 请将所有介质预热到37° c。
<ol><li>
小鼠胚胎成纤维细胞 (MEF) 培养基 (准备500毫升)
	<ol>
<li>添加50毫升胎牛血清 (FBS) 到449毫升 Dulbecco 氏改良鹰的培养基 (DMEM), 并通过500毫升0.22 µm 孔过滤器过滤器。</li>
		<li>在过滤后加入1毫升抗菌剂 (50 毫克/毫升)。存储在4° c 1 月或分的媒体和存储在≤-20 ° c。</li>
	</ol>
</li>
	<li>
N2 介质 (用于准备500毫升)
	<ol>
<li>添加5毫升 l-丙氨酸-l-谷氨酰胺 (100x) 和500µL 2-基 (55 mM) 至489.5 毫升 DMEM: 营养混合物 F-12 (DMEM/F-12), 并通过500毫升0.22 µm 孔过滤器过滤器。</li>
		<li>在过滤后加入1毫升抗菌剂 (50 毫克/毫升) 和5毫升 N2 补充物。将介质存储在4° c 的黑暗中长达1月。</li>
	</ol>
</li>
	<li>
无血清生长培养基 (KSR) (用于制备500毫升)
	<ol>
<li>添加75毫升无血清培养基补充剂, 5 毫升 l-谷氨酰胺 (100x), 5 毫升记忆非必需氨基酸 (NEAA) (100x), 和500µL 2-基 (55 毫米) 到413.5 毫升非谷氨酰胺含有 DMEM, 并通过500毫升0.22 µm 孔过滤器单位过滤。</li>
		<li>在过滤后加入1毫升抗菌剂 (50 毫克/毫升)。将介质存储在4° c, 最多可达1月。</li>
	</ol>
</li>
	<li>
小鼠胚胎干细胞培养基 (用于制备500毫升)
	<ol>
<li>添加75毫升干细胞级 FBS, 5 毫升记忆 NEAA (100x), 5 毫升 l-谷氨酰胺 (100x), 500 µL 2-基 (55 毫米) 至413.5 毫升非谷氨酰胺含有 DMEM, 并通过500毫升0.22 µm 孔过滤器单位过滤。</li>
		<li>在过滤后加入1毫升抗菌剂 (50 毫克/毫升)。存储在4° c 在黑暗中的媒体长达1月或分和存储在≤-20 ° c。</li>
	</ol>
</li>
</ol>2. 培养 mESCs
<ol><li>分裂将 mef 介质中的不活泼的 mef 馈电单元放到组织培养处理的板上 3-4 x 104单元格/cm2。允许至少12小时, 让他们在37° c 与≥95% 相对湿度和 5% CO2前的细胞培养孵化器中沉淀 mESCs。如果不立即使用, 请每3天更换一次 MEF 介质。在电镀后7天内不使用 MEFs 处理。</li>
	<li>在含有小鼠白血病抑制因子 (mLIF) (1000 U/毫升) 的制介质中添加 mESCs (密度范围从 1-4 x 104单元格/cm2) 到 MEF 板。孵育的细胞在37° c 与≥95% 相对湿度和 5% CO2。</li>
	<li>mESCs 使用胰蛋白酶-EDTA (0.05% 胰蛋白酶) (1:5-1:10) 一旦菜变成 ~ 70-80% 汇合或如果殖民地开始接触。这通常发生每2天, 但可能需要多达4或5天取决于初始电镀密度。</li>
	<li>保持 mESCs 在制介质中的 MEF 馈线层上保持多。</li>
	<li>
在开始分化前, 将细胞至少一次涂在明胶涂层板上, 而不 MEFs 稀释 MEFs。
	<ol>
<li>在磷酸盐缓冲盐水 (PBS) 中加入0.1% 明胶, 用钙2 +/毫克2 +对组织培养处理皿, 在37° c 下至少1小时, 制备明胶涂层板. 板 1.5-2 x 106 cells/10 cm 板 (2.7-3.6 x 104单元格/cm2), 并允许单元格在开始分化前2天展开。</li>
	</ol>
</li>
</ol>3. 区分 mESCs 向 MGE 样 Telencephalic 祖
<ol><li>
分化日 (DD) 0 = "浮细胞"
	<ol>
<li>mESCs 在明胶涂层板上生长2天后, 在没有 Ca2 +/Mg2 +的情况下, 用 PBS 吸吸介质并冲洗一次细胞。添加足够的胰蛋白酶-edta (0.05% 胰蛋白酶) 覆盖板的表面 (通常4毫升胰蛋白酶-edta 为一个 10 cm 组织培养皿), 并把细胞回到孵化器在37° c 与≥相对湿度和 5% CO2 4 分钟。</li>
		<li>4分钟后, 用2倍于制介质的体积来淬火胰蛋白酶-EDTA。将电池转移到适当大小的离心管上, 并将细胞在 200 x g 处离心5分钟。5分钟后, 取出导管, 吸出介质而不干扰颗粒。重1毫升 KSR:N2 介质 (1:1) 中含有 BMP 抑制剂 LDN-193189 (250 nM) 和 Wnt 抑制剂 XAV-939 (10 µM) 的颗粒。</li>
		<li>使用例或自动电池计数器测量细胞浓度。在非附着的组织培养皿中, 在含有 LDN-193189 (250 nM) 和 XAV-939 (10 µM) 的 KSR:N2 培养基 (1:1) 中加入7.5万细胞/毫升, 以作为胚状体 (EBs) 开始生长细胞。孵育的细胞在37° c 与≥95% 相对湿度和 5% CO2。</li>
	</ol>
</li>
	<li>在 DD1, 用多聚 l-赖氨酸 (10 µg/毫升) 在 PBS 中处理的细胞 "着陆", 在37° c (2 +/毫克2 +) 上过夜 (O/N), 以≥95% 相对湿度或至少 1 h。</li>
	<li>在 DD2 上, 吸出多聚 l-赖氨酸, 并在 PBS 中涂上层粘连蛋白 (10 µg/毫升, Ca2 +/毫克2 +) O/N 在37° c 时具有≥95% 相对湿度。 注意: 虽然 O/N 是最优的, 但仅有2小时就足够了。如果第二天没有使用板, 抽吸层粘连蛋白, 在没有 Ca2 +/Mg2 +的情况下更换 PBS, 并在4° c 存储2周。</li>
	<li>
DD3 ("土地细胞")
	<ol>
<li>在开始 EB 离解之前, 吸取层粘连蛋白, 并让板块完全干燥在一个组织培养罩。不要使用看起来有光泽或明显潮湿的盘子。将 ebs 与介质传输到15毫升的管中, 并将其离心3-4 分钟, 在 15 x g 或 ebs 有颗粒状。</li>
	</ol>
</li>
	<li>吸气培养基, 并加入3毫升的细胞剥离液含有 dnasei (2 U/毫升) 和孵化在37° c 与≥95% 相对湿度和 5% CO2为15分钟. 轻轻地弹管每3分钟, 以协助 EB 离解。</li>
	<li>一旦 EBs 不再可见或15分钟已经过去了, 添加6毫升 KSR:N2 (1:1) 包含 dnasei (1 U/毫升) 和离心机为5分钟 200 x g。</li>
	<li>将 KSR:N2 (1:1) 中含有 LDN-193189 (250 nM)、XAV-939 (10 µM) 和岩石抑制剂 Y-27632 (10 µM) 的细胞在 4.5-5 x 104单元格/cm2中板化。</li>
</ol>4. SST 丰富的议定书: "DD12 GFP 高声波刺猬 (嘘)" (继续从步骤 3.7)
<ol><li>在 DD5 上, 改变媒体与 KSR:N2 (1:1) 包含 FGF-2 (10 ng/毫升), IGF-1 (20 ng/毫升), 和 SSH (50 ng/毫升)。</li>
	<li>使用步骤 3.2-3.3 中概述的说明, 准备8天 (步骤 4.4) 重新电镀组织培养皿。</li>
	<li>在 DD7 上, 改变媒体与 KSR:N2 (1:1) 含有 FGF-2 (10 ng/毫升), IGF-1 (20 ng/毫升), 和嘘 (50 ng/毫升)。</li>
	<li>
在 DD8 上, 重新板细胞。 注意: 虽然这一步是不必要的, 重新电镀的细胞可以减少早期区分, 不需要的细胞类型。再电镀也会分解细胞的球, 使得下游的免疫组化分析变得困难, 并在以后的时间点提高了外地资产管制中心的效率。<ol>
<li>重新板细胞, 分离细胞从板与胰蛋白酶-EDTA (0.05% 胰蛋白酶) 为 5 min 在37° c 以≥相对湿度和 5% CO2。用2倍于 KSR:N2 的体积将胰蛋白酶-EDTA 淬火, 并在 200 x g 上离心5分钟. 通过40µm 滤筒过滤细胞, 去除任何块状。在含有 FGF-2 (10 ng/毫升)、IGF-1 (20 ng/毫升) 和 Y-27632 (10 µM) 和嘘 (50 ng/毫升) 的 N2/KSR (1:1) 中重新将细胞在25万细胞/cm2 。 注意: 如果相反, 决定不重新板块的细胞在 DD8, 改变媒体与 KSR:N2 包含嘘 (50 ng/毫升) 每2天从 DD7 到 DD12, 并继续添加 IGF-1 (20 ng/毫升) 和 FGF-2 (10 ng/毫升), 直到 DD9。</li>
	</ol>
</li>
	<li>在 DD10 上, 改变媒体与 KSR:N2 包含嘘 (50 ng/毫升)。</li>
	<li>
在 DD12 上, 要获得富含 SST 亚型的细胞, 请使用外地资产管制室来隔离 Lhx6::GFP-only 表达细胞。
	<ol>
<li>要分离细胞, 使用非胰蛋白酶含有细胞离解试剂, 而不是胰蛋白酶-EDTA。使用 PBS 清洗细胞一次, 不需要 Ca2 +/Mg2 +。在细胞中加入含有 dnasei (2 U/毫升) 的前温热的非胰蛋白酶含有细胞离解试剂。将它们放在37° c 的恒温箱中, ≥相对湿度和 5% CO2用于10-30 分钟或直到细胞分离。每隔5分钟轻轻拍打盘子, 以助解离。 注: 对于 DD11-12 文化, 只有10-15 分钟可能是必要的。对于 DD15-16 文化, 30 分钟或以上可能需要完全离解。</li>
		<li>一旦细胞完全解除了板块, 继续使用标准的协议或使用其他下游分析中的单元格隔离。</li>
	</ol>
</li>
</ol>5. 光伏富集协议: "DD11/DD17 mCherry + aPKCi" (从步骤3.7 延续)
<ol><li>在 DD5 上, 改变 KSR:N2 (1:1) 含有 FGF-2 (10 ng/毫升) 和 IGF-1 (20 ng/毫升) 的介质。</li>
	<li>使用步骤 3.2-3.3 中概述的说明, 准备 DD8 (步骤 4.4) 的组织培养板进行重新电镀。</li>
	<li>在 DD7 上, 改变 KSR:N2 (1:1) 含有 FGF-2 (10 ng/毫升) 和 IGF-1 (20 ng/毫升) 的介质。</li>
	<li>在 DD8 上, 将步骤4.4 中所述的单元格重新装入, 但有以下例外情况: 当重新电镀细胞时, 用快速激动剂 (凹陷; 30 nM) 和添加不典型 PKC 抑制剂 (aPKCi; 2 µM) 来代替嘘。两者合计, 再电镀介质现在 N2/KSR (1:1) 含有 FGF-2 (10 ng/毫升), IGF-1 (20 ng/毫升), Y-27632 (10 µM), 凹陷 (30 nM), 和 aPKCi (2 µM)。</li>
	<li>在 DD10 上, 用含有 aPKCi (2 µM) 的 KSR:N2 改变介质。</li>
	<li>
DD11 注: 在这一点上, Nkx2.1:: mCherry + 细胞富集成为光伏 CIns。<ol>
<li>使用步骤4.6 中概述的相同协议将单元格从盘中分离出来。如果作出决定收集 Nkx2.1:: mCherry + 细胞在以后的分化日, 无视这一步。</li>
	</ol>
</li>
	<li>在 DD12 上, 用含有 aPKCi (2 µM) 的 KSR:N2 改变介质。在 DD14 上, 用含有 aPKCi (2 µM) 的 KSR:N2 改变介质。在 DD16 上, 用含有 aPKCi (2 µM) 的 KSR:N2 改变介质。在 DD17 上, 收集 Nkx2.1:: mCherry + 使用步骤4.6 中概述的相同协议的单元格。</li>
</ol>6. 光伏富集协议: "DD17 mCherry 低嘘" (从步骤3.7 延续)
<ol><li>在 DD5 上, 改变 KSR:N2 (1:1) 含有 FGF-2 (10 ng/毫升) 和 IGF-1 (20 ng/毫升) 的介质。在 DD7 上, 改变 KSR:N2 (1:1) 含有 FGF-2 (10 ng/毫升) 和 IGF-1 (20 ng/毫升) 的介质。</li>
	<li>
在 DD9 上, 使用 KSR:N2 (1:1) 更改介质。从这一点开始, 不需要额外的增长因素。
	<ol>
<li>继续每2天更改一次媒体, 直到在 DD17 上捕获细胞。</li>
	</ol>
</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+versus+Basal+Neurogenesis+Directs+Cortical+Interneuron+Subclass+Fate.">Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate.</a> Cell Rep. 13, 1090-1095 (2015).</li><li>Xu, Q., Tam, M., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+Nkx2.1-lineage+cells+in+the+mouse+telencephalon.">Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.</a> J Comp Neurol. 506, 16-29 (2008).</li><li>Wonders, C. P., 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+spatial+bias+for+the+origins+of+interneuron+subgroups+within+the+medial+ganglionic+eminence.">A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence.</a> Dev Biol. 314, 127-136 (2008).</li><li>Inan, M., Welagen, J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+bias+in+the+mitotic+origins+of+somatostatin-+and+parvalbumin-expressing+interneuron+subgroups+and+the+chandelier+subtype+in+the+medial+ganglionic+eminence.">Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.</a> Cereb Cortex. 22, 820-827 (2012).</li><li>Flames, N., 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=Delineation+of+multiple+subpallial+progenitor+domains+by+the+combinatorial+expression+of+transcriptional+codes.">Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 27, 9682-9695 (2007).</li><li>Fogarty, 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=Spatial+genetic+patterning+of+the+embryonic+neuroepithelium+generates+GABAergic+interneuron+diversity+in+the+adult+cortex.">Spatial genetic patterning of the embryonic neuroepithelium generates GABAergic interneuron diversity in the adult cortex.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 10935-10946 (2007).</li><li>Eiraku, 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=Self-organized+formation+of+polarized+cortical+tissues+from+ESCs+and+its+active+manipulation+by+extrinsic+signals.">Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.</a> Cell Stem Cell. 3, 519-532 (2008).</li><li>Colasante, G., 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=Rapid+Conversion+of+Fibroblasts+into+Functional+Forebrain+GABAergic+Interneurons+by+Direct+Genetic+Reprogramming.">Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming.</a> Cell Stem Cell. 17, 719-734 (2015).</li><li>Xu, Q., Cobos, I., De La Cruz, E., Rubenstein, J. 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作者没有什么可透露的。

虽然这种方法是非常有效的模式 J1-derived mESCs (SCRC-1010), 我们已经经历了可变的成功与其他制线和克隆分离株。例如, Foxg1::venus mESCs (EB3-derived;Danjo et al.13) 对此协议的响应很差, DD12 的 Foxg1 归纳通常按1-2% 的顺序进行。出于我们不完全理解的原因, 另一个 Nkx2.1:: mCherry: Lhx6:: GFP 双报告克隆 (称为 JQ59), 同时被隔离为该协议中描述的线 (称为 JQ27), 生长时产生少于 1% Nkx2.1 表达的细...

在两个小鼠和人类中, 大约一半的皮质抑制神经元 (CIns) 起源于一个短暂的皮层下结构, 称为内侧节隆起 (MGE), 上皮祖和其他神经元和神经胶质子组表示转录因子 Nkx2.11,2。通过相交形态学、神经、电生理学和连通性特征3,4来定义这些子类型。MGE 衍生的 CIns 可以分为大多数非重叠子群的基础上, 其表达的 PV 或 SST, 其表达与特定的电生理和连接倾向5。神经元, 特别是 PV 亚群的功能障碍, 已牵连多神经精神疾病和疾病6,7。该方法的总目标是产生干细胞衍生的有丝分裂祖细胞和迁移前体, 丰富的 PV 或 SST 的命运, 研究皮质 interneuron 生物学和用于细胞基础治疗。
我们已经开发了一个可伸缩的, 高效的方法来推导 Nkx2.1 表达 interneuron 祖及其后代从 mESCs。使用 Nkx2.1:: mCherry: Lhx6:: GFP 双报告制线8, Nkx2.1 祖细胞或其 Lhx6-expressing 后有丝分裂后代可通过外地资产管制系统隔离, 随后用于若干下游应用。通过操纵一些信号通路、培养时间和神经再生模式, 我们可以获得数百万个荧光标记的 interneuron 前体, 适合于下游应用的宿主。
虽然存在一些其他方法来生成 MGE 样的祖 mESCs9,10,11,12,13,14, 我们的方法, 这依赖于 Wnt拮抗剂 XAV-939, 是特别有效的产生 Foxg1/Nkx2.1 co 表达 telencephalic 祖细胞。此外, 通过我们的双报告系统选择 interneuron 祖细胞或其后有丝分裂 Lhx6-expressing 后代的能力, 极大地提高了产生不同祖和后代的能力。

<table><tbody><tr><td>Bottle-top vacuum filter system</td><td>Corning</td><td>CLS430769</td><td></td></tr><tr><td>Test Tube with Cell Strainer Snap Cap</td><td>ThermoFisher</td><td>Corning 352235</td><td></td></tr><tr><td>Mouse embryonic fibroblasts (CF-1 MEF IRR 7M)</td><td>MTI-Globalstem</td><td>GSC-6101G</td><td>1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: &lt;sup class=&quot;xref&quot;&gt;29&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;35&lt;/sup&gt;</td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150H</td><td></td></tr><tr><td>Primocin</td><td>Invivogen</td><td>Ant-pm-2</td><td>Also known as antimicrobial agent. Do not filter with base media -- add after filtration. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;36&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;37&lt;/sup&gt;</td></tr><tr><td>N2 supplement-B</td><td>Stemcell Technologies</td><td>7156</td><td>Do not filter with base media -- add after filtration</td></tr><tr><td>Glutamax (100x)</td><td>ThermoFisher</td><td>35050061</td><td>Also known as L-alanine-L-glutamine. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;38&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;39&lt;/sup&gt;</td></tr><tr><td>KnockOut Serum Replacement (KSR)</td><td>ThermoFisher</td><td>10828028</td><td>Also known as serum-free medium supplement. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>L-glutamine (100x)</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>ThermoFisher</td><td>11140050</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>ThermoFisher</td><td>21985023</td><td></td></tr><tr><td>KnockOut DMEM</td><td>ThermoFisher</td><td>10829018</td><td>Also known as non-glutamine containing DMEM. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>Hyclone FBS</td><td>VWR</td><td>82013-578</td><td>Also known as stem cell grade FBS. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>Tissue culture treated dish (10cm)</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Non-adherent sterile petri dish (10cm)</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>Leukemia inhibitory factor (mLIF)</td><td>Chemicon</td><td>ESG1107</td><td>Do not freeze, store at 4&#039;C. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>DMEM/F12</td><td>ThermoFisher</td><td>11330032</td><td></td></tr><tr><td>0.1% Gelatin Solution</td><td>ATCC</td><td>ATCC PCS-999-027</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P6282</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Accutase</td><td>ThermoFisher</td><td>A1110501</td><td>Also known as non-trypsin containing cell dissociation reagent. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>RQ1 RNase-Free DNase</td><td>Promega</td><td>M610A</td><td></td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074</td><td>Resuspend in DMSO and store at -80&#039;C in single use aliquots</td></tr><tr><td>XAV939</td><td>Stemgent</td><td>04-0046</td><td>Resuspend in DMSO and store at -80&#039;C in single use aliquots</td></tr><tr><td>rhFGF-2</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;C in single use aliquots</td></tr><tr><td>rhIGF-2</td><td>R&amp;amp;D Systems</td><td>291-G1</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;C in single use aliquots</td></tr><tr><td>ROCK inhibitor (Y-27632)</td><td>Tocris</td><td>1254</td><td>Resuspend in DMSO and store at -80&#039;C in single use aliquots</td></tr><tr><td>Smoothened agonist (SAG)</td><td>Millipore</td><td>566660-1MG</td><td>Resuspend in H20 and store at -80&#039;C in single use aliquots</td></tr><tr><td>rm Sonic Hedgehog/SHH</td><td>R&amp;amp;D Systems</td><td>464-SH-025</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;C in single use aliquots</td></tr><tr><td>PKC&amp;zeta; Pseudosubstrate Inhibitor, Myristoylated</td><td>EMD Millipore</td><td>539624</td><td>Resuspend in H20 and store at -80&#039;C in single use aliquots</td></tr></tbody></table>

differentiation of mouse embryonic stem cells into cortical interneuron precursors

gaba 皮质神经元是一种异构的细胞群体, 在调节兴奋性锥体神经元的输出以及同步锥体神经元集合的输出方面起着关键作用。interneuron 功能的缺陷已经牵连到各种神经精神疾病, 包括精神分裂症, 自闭症和癫痫。从胚胎干细胞衍生的皮质神经元不仅允许研究其发展和功能, 但提供了深入的分子机制的基础上, 皮层 interneuron 相关疾病的发病机理。神经元也有非凡的能力, 生存, 迁移, 并集成到宿主皮质电路后移植, 使他们成为理想的候选细胞的治疗方法。在这里, 我们提出了一个可伸缩, 高效, 改良的胚体的单层方法, 以 Nkx2.1 表达 interneuron 祖细胞和他们的后代从小鼠胚胎干细胞 (mESCs)。使用 Nkx2.1:: mCherry: Lhx6:: GFP 双报告制线、Nkx2.1 祖细胞或其 Lhx6-expressing 后有丝分裂后代, 可通过荧光活化单元分类 (资产管制系统) 隔离, 随后用于许多下游应用。我们还为发育 (PV) 或生长抑素 (SST) interneuron 亚群提供了丰富的方法, 这可能有助于研究命运的确定, 或用于治疗应用, 将受益于 interneuron 亚组丰富移植.

本文所描述的协议是我们已发布协议的修改版本15,16,17 , 并已针对我们的 Nkx2.1 进行了优化:: mCherry: Lhx6:: GFP 双报告制线。通过添加 Wnt 抑制剂 XAV-939 从 DD0-5, 结合重新电镀在 DD8, 我们实现了鲁棒 Nkx2.1 诱导, 其中50% 以上的 DAPI + 核在文化中也 Nkx2.1 表达 (图 1A, B)。免疫组...

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Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

本议定书描述了一种方法, 产生皮层 interneuron 祖细胞和后有丝分裂 interneuron 前体从小鼠胚胎干细胞使用改良的胚体-单层法。这些祖/前体可用于体外或荧光分类和移植到新生儿大脑皮层研究命运确定, 或用于治疗应用。

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 ...

differentiation-mouse-embryonic-stem-cells-into-cortical-interneuron

Research

JoVE Journal

Neuroscience

10.3K 次观看.  University of Pennsylvania. 本议定书描述了一种方法, 产生皮层 interneuron 祖细胞和后有丝分裂 interneuron 前体从小鼠胚胎干细胞使用改良的胚体-单层法。这些祖/前体可用于体外或荧光分类和移植到新生儿大脑皮层研究命运确定, 或用于治疗应用。

视频: Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

科研

生物学

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 ...

发育生物学

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

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

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 of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

神经科学

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 Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://app.jove.com/v/56358">Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors.</a> J. Vis. Exp. (2017)
This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

从小鼠胚胎干细胞产生皮质中间神经元前体

1. Media Preparation
NOTE: Warm all media to 37 &#38;#176;C before use in cell culture.

	Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	
	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

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/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 ...

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://app.jove.com/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

将小鼠胚胎干细胞分化为联网神经元群

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...