We are grateful for the contributions of Yolanda Tafuri in optimizing conditions for SH-SY5Y differentiation, and for the support of Dr. Lynn Enquist, in whose lab this work was initiated. Y. Tafuri contributed the images shown in Figure 3. This work was supported by the NIH-NIAID Virus Pathogens Resource (ViPR) Bioinformatics Resource Center (MLS and L. Enquist) and K22 AI095384 (MLS).

1.一般注意事项<ol><li> 见材料/设备的表所需试剂的列表。执行严格的无菌条件下的所有步骤。 </li><li>请对所有媒体的准备工作，包括FBS，热灭活的胎牛血清（hiFBS）。对热灭活，温暖的50ml等分试样的FBS在56℃下进行30分钟，倒相，每10分钟（也见表 1）。 注意:当FBS的是无热灭活所使用的，上皮样表型在整个SH-SY5Y细胞的培养物更快速地进展。 </li><li>在使用之前，允许媒体温热并在培养箱平衡以建立每一步之前一个适当的pH值的平衡。例如，将50ml的媒体花费大约一小时，以充分平衡（pH为7，在37℃，5％CO 2）。 注意:此协议使用，需要部分分化的SH-SY5Y细胞中胰蛋白酶处理并重新铺板的两步分裂过程。这是一个紧张这些异常脆弱的细胞过程。因此，孵育在胰蛋白酶的细胞的时间最小量是重要的。这将允许对神经元的优先剥离，留下仍然附着于培养皿的上皮样细胞。 </li><li>执行分化的细胞的研磨，用10毫升的塑料吸管与针对包含该细胞的锥形管的底部尖端缓慢。以缓慢的速度进行研磨，上下不超过五次。 </li></ol> 2. SH-SY5Y维护文化的通道<ol><li>拆分维护文化当细胞达到70-80％汇合，和不超过10〜15代。文化通常都需要每3-5天传代（假设文化分裂过程中，不会比稀释5倍以上）。 </li><li>到从T-75烧瓶中传代细胞，吸媒介，然后用大约10mL，1×PBS中冲洗。 注意:我们不建议分裂SH-SY5Y维护文化福rther比1:传代过程中5，因为这会导致细胞死亡，由于低汇合。 </li><li>抽吸PBS，然后添加2.5毫升0.05％胰蛋白酶-EDTA（1×）。 </li><li>孵育在培养箱2-3分钟，尖轻轻从烧瓶的表面释放细胞。 </li><li>加入10 mL的基本生长培养基（ 见表 2）和磨碎的1-2倍。 </li><li>降速2分钟在1000 XG，抽吸介质，然后悬浮在5毫升的基本生长培养基沉淀。 </li><li>稀释细胞从1:3至1:5的20毫升正常电镀中的T-75烧瓶中的总体积，或对分化（第5部分）计算和板。 </li></ol> 3.冷冻SH-SY5Y细胞<ol><li>冻结补充有5％（体积/体积）的DMSO中的基本生长介质的SH-SY5Y成神经细胞瘤细胞的早期传代。 </li><li>最初，在-80℃冷冻等分试样24小时，然后转移到液氮长期贮存。 注意:作为参考，汇合（75-85％），T-75瓶将产生5SH-SY5Y细胞的1ml等份冷冻。每个等分试样的应包含任何地方从2-5百万细胞总。 </li></ol> 4.解冻和文化未分化的SH-SY5Y神经母细胞瘤<ol><li>准备基本生长介质。 </li><li>迅速解冻冷冻的细胞在37℃水浴（约2分钟）。 </li><li>重悬细胞于9ml基本生长培养基在15ml锥形管中，然后离心以1000xg 2分钟。 </li><li>吸出上清液同时小心不要打扰沉淀的细胞，并轻轻悬浮细胞在10毫升的基本生长介质。 </li><li>板细胞到T-25瓶或60 平方毫米的菜。 </li><li>次日，更换培养基，以去除死细胞。 </li></ol> 5. 0日:分化细胞电镀<ol><li> 参见图1分化时间表。 </li><li>冲洗未分化细胞用1×PBS，吸出物，然后trypsinize用1-2毫升温热1×0.05％胰蛋白酶-EDTA。 </li><li>当细胞在胰蛋白酶，孵育在培养箱约3分钟。 </li><li>通过加入10ml的基本生长培养基猝灭胰蛋白酶，冲洗烧瓶或盘的两侧，并轻轻磨碎1-3次。传送内容到15毫升锥形管中。 </li><li>离心机在1000 XG 2分钟，吸媒体同时小心不要打扰颗粒。 </li><li>在5毫升的基本生长培养基重悬沉淀和磨碎1-3次。 </li><li>算使用血球细胞，然后稀释使用基本生长培养基至50,000个细胞/ ml。 </li><li>板2萃取池的，有35 平方毫米的菜，共计每盘100,000个细胞，并放回孵化器。 </li></ol> 6.第1天:改变传媒（分化媒体＃1） <ol><li>分装50毫升分化媒体＃1（ 见表 2），并在37℃水浴孵育。 </li><li>当媒体被加热，允许它在一个孵化器平衡（37℃，5％CO 2）至少1小时，以建立在使用前一个适当的pH值的平衡。 </li><li>添加视黄酸（RA）（见表1），以温热并平衡媒体立即将介质添加到菜之前。 注意:视黄酸是光敏感，应在4℃下贮存在黑暗的瓶子</li><li>轻轻吸了旧的媒体和丢弃。 </li><li>加2ml分化媒体＃1？35 平方毫米的菜RA，并返回到孵化器。 </li></ol> 7.第3天:改变传媒（分化媒体＃1） <ol><li>重复第6（1-5步） </li></ol> 8.第5天:改变传媒（分化媒体＃1） <ol><li>重复第6（1-5步） </li></ol> 9日7:拆分单元格1:1 <ol><li>添加到RA温暖和平衡的分化媒体＃1，立即将介质添加到菜肴之前。 </li><li>轻轻吸了旧的媒体和丢弃。 </li><li>加入200μl温热0.05％1×胰蛋白酶的EDTA？35 平方毫米培养皿和温暖培养箱大约2-3分钟，或直至细胞被明显地从显微镜下观察到的板抬起。 </li><li>通过，有35 平方毫米的菜加入2ml分化媒体＃1 RA淬火胰蛋白酶 并利用媒体来冲洗剩余的神经细胞脱盘。然后内容物转移到50ml锥形管中。 注:在胰蛋白酶消化步骤，不要一下子trypsinize太多的菜肴。这有助于确保神经元培养物中没有胰蛋白酶温育的时间过长，它可以是细胞毒性的。 </li><li>从多达10个菜在50ml锥形管中结合内容并轻轻慢慢磨碎上下不超过五次，用10毫升的塑料吸管。 </li><li>分装2 ml细胞悬液注入新鲜35毫米2的菜肴，并返回到孵化器。 </li></ol> 10.第8天:改变传媒（分化媒体＃2） <ol><li>加入RA（见&lt;STRONG&gt;表1），以温暖，并立即将介质添加到菜肴之前平衡媒体。 </li><li>轻轻吸了旧的媒体和丢弃。 </li><li>慢慢加入2毫升分化媒体＃2（ 见表 2） 有，有35 平方毫米的菜，并回归到孵化器RA。不允许的神经元被暴露于空气的时间，因为它们能快速变干较长的时间。 </li></ol> 11.第9天:准备细胞外基质（ECM）包被的培养皿<ol><li>在冰上解冻的ECM解决方案一小瓶，稀释1:100到寒冷的DMEM。 </li><li>分配2毫升混合物成35个平方毫米培养皿，并确保了培养皿的整个基被覆盖。 </li><li>在培养箱地方（37℃，5％CO 2）1小时或过夜。 </li><li>抽吸混合物和允许空气干燥在发动机罩约1小时。在室温下储存长达2个月。 </li></ol> 12.第10天:细胞转移到ECM涂层板1:1 <ol><li>加入RA（ 见表 1），以温暖和平衡媒体立即将介质添加到菜肴之前。 </li><li>轻轻吸了媒体和丢弃。 </li><li>加入200μl温热胰蛋白酶至35 平方毫米培养皿，并允许在室温下孵育约1-2分钟，或直至神经元被明显地从显微镜下观察到的培养皿解除。 注:执行在室温下此胰蛋白酶处理步骤，以免过度孵育用胰蛋白酶的神经元并导致损坏。从神经元比板块在这个阶段上皮样细胞更快释放。 </li><li>通过加入2ml分化媒体＃2？35 平方毫米的菜淬火胰蛋白酶和利用媒体来冲洗剩余的神经细胞脱盘。然后内容物转移到50ml锥形管中。 </li><li>从多达10个菜在50ml锥形管中结合内容并轻轻慢慢磨碎上下不超过五次10毫升的塑料吸管。 </li><li>免除2 ml细胞悬液注入ECM-涂35毫米2的菜肴，并返回到孵化器。 </li></ol> 13.第11天:改变传媒（分化传媒＃3） <ol><li>加入RA（ 见表 1），以温暖和平衡媒体立即将介质添加到菜肴之前。 </li><li>轻轻吸了旧的媒体和丢弃。 </li><li>慢慢RA加2ml分化传媒＃3（ 见表 2），有35 平方毫米的菜，并返回到孵化器。不允许的神经元被暴露于空气的时间延长的时间。 </li></ol> 14.第14天:改变传媒（分化传媒＃3） <ol><li>重复第13（第1-3步） </li></ol> 15. 17日:最近媒体更改（分化传媒＃3） <ol><li>重复第13（第1-3步） </li></ol> 16. 18日:神经细胞即用<ol><li>更改媒体鲜DIFFerentiation媒体＃3 RA每3天来维持神经元的健康。 注:细胞应分化成神经元和表现出神经元表型。培养通常为以下终末分化长达14天的稳定，但是神经元存活率的持续时间取决于在分化开始时的未分化细胞的传代次数。较高传代数得到具有较短的使用寿命分化的神经元。 </li></ol>

The authors have nothing to disclose.

上述协议提供了一个直接的和可重复的方法来生成均匀的和可行的人类神经文化。该协议利用技术和集成多个先前公布的方法1-4和目标划定每个最佳实践做法。 SH-SY5Y细胞的分化依赖于逐渐血清剥夺;加入视黄酸，神经营养因子和细胞外基质蛋白;和串行分裂来选择分化成熟的神经细胞贴壁。该细胞系开始以贴壁和悬浮的细胞的异质群体。该协议的目的是通过传代或媒体变更前预提PBS洗涤以?...

体外模型系统中使用的能力已大大增强神经生物学和神经科学领域。培养中的细胞提供了一个高效的平台来表征蛋白质的功能和分子机制的具体现象，了解疾病和感染的病理，并进行初步的药物测试评估。在神经生物学，主要类型的细胞培养模型包括例如大鼠B35细胞5，神经2A小鼠细胞6和大鼠PC12细胞7从大鼠和小鼠，以及成神经细胞瘤细 ​​胞系的原代神经元培养物。虽然使用这样的细胞系的已显著先进领域，有与处理非人类细胞和组织相关的几个混杂因素。相比于人体时，这些包括理解特定物种的代谢过程的差异，疾病的表现，和发病机制的表型。同样重要的是要注意，再是小鼠和人的基因表达和转录因子信号之间显著差异，突出啮齿动物模型的局限性和谅解其途径是啮齿动物和人类8-11之间是保守的重要性。其他已经采用了利用人体的神经元细胞系，包括N型的Tera-2（NT2）人畸胎瘤细胞和诱导多能干细胞（iPS细胞）的。这些细胞系为体外人类系统不错的机型。然而，NT2细胞用视黄酸（RA）的结果的神经元，星形胶质细胞的混合群的产生，和径向胶质细胞12，因此需要另外的纯化步骤的分化得到的神经元的纯群体。此外，NT2细胞表现出高度可变的核型13，具有在细胞的72％大于60染色体。 iPS细胞表现出变异性在不同细胞系之间的差异，并在分化效率变化<html> 14，它因此希望具有一个一致的和可重复的人神经元细胞模型，以补充这些替代。 SH-SY5Y成神经细胞样细胞是亲神经母细胞瘤细胞系SK-N-SH的亚克隆。从包含两个成神经细胞样细胞和上皮样细胞15骨髓活检于1970年产生的亲本细胞系。 SH-SY5Y细胞具有由47条染色体的稳定核型，并且可以从一个神经细胞样状态成成熟的人神经元，通过各种不同的机制，包括使用的RA，佛波酯和特定神经营养蛋白诸如脑源性分化神经营养因子（BDNF）。现有的证据表明，使用不同的方法，可以为特定的神经元亚型如肾上腺素，胆碱，和多巴胺能神经元16,17选择。这后一方面使得的神经生物学实验的大量有用的SH-SY5Y细胞。 内容】"&gt;几项研究已经注意到在其未分化和分化的状态SH-SY5Y细胞之间的重要区别。当SH-SY5Y细胞是未分化的，它们迅速增殖并似乎非偏振光，除了极少数，短流程。它们通常生长表示未成熟的神经元18,19的中块和表达的标志物。当有区别的，这些细胞扩展长的，支链的过程，减少增殖，并且在一些情况下，极化2,18。完全分化的SH-SY5Y细胞先前已经证明表达多种成熟神经元包括生长相关蛋白（GAP-43），神经元核（的NeuN），突触素（SYN），突触小泡蛋白II（SV2），神经元特异性烯醇化酶（NSE）和微管相关蛋白（MAP）的不同的标记的2,16,17,20，和缺乏神经胶质标记物，如胶质纤维酸性蛋白（GFAP）4的表达。在进一步的支持，分化SH-SY5Y细胞represeNT均匀的神经元群，拆除BDNF的导致细胞凋亡4。这表明，分化SH-SY5Y细胞的存活依赖于营养因子，类似于成熟神经元。 SH-SY5Y细胞的使用增加了，因为亚克隆成立于1978年3。它们的使用的一些实例包括:调查帕金森氏病17，阿尔茨海默氏病21，和病毒感染，包括脊髓灰质炎病毒22，肠道病毒71型的发病23,24 ，水痘带状疱疹病毒（VZV）1，人巨细胞病毒25，和单纯疱疹病毒（HSV）2,26。使用SH-SY5Y细胞在他们的未分化的形式使用这些细胞要注意，一些研究，特别是在neurovirology 27-36领域是很重要的。在的未分化与分化的SH-SY5Y细胞中观察到的表型的差异引起的磨片的问题疗法感染的观察进展会在成熟分化的神经元的不同。例如，分化的SH-SY5Y细胞具有HSV-1的摄取与未分化，增殖SH-SY5Y细胞，这可能是因为缺乏结合HSV并调节上未分化的SH-SY5Y细胞2条目表面受体的一个更高的效率。因此，关键是设计侧重于测试在体外的神经元的实验时，SH-SY5Y细胞应以获得翻译和对比的最准确的结果，以在体内模型区分。 一个可靠的方法的发展，产生人类神经细胞当务之急是让研究人员进行翻译实验证明，精确地模拟人的神经系统。这里介绍的协议是界定从以前的方法得出1-4最佳实践来丰富对于那些分化的人神经过程使用视黄酸。

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">B-27</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:133px;">17504-044</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:141px;">Brain-Derived Neurotrophic Factor (BDNF)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">SRP3014 (10ug)/B3795 (5ug)</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">dibutyryl cyclic AMP (db-cAMP)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">D0627</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">DMSO</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:133px;">4-X</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:141px;">Minimum Essential Medium Eagle (EMEM)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">M5650</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:141px;">Hyclone</td>
			<td style="width:133px;">SH30071.03</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">GlutamaxI</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">35050-061</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Glutamine</td>
			<td style="width:141px;">Hyclone</td>
			<td style="width:133px;">SH30034.01</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Potassium Chloride (KCl)</td>
			<td style="width:141px;">Fisher Scientific</td>
			<td style="width:133px;">BP366-1</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:141px;">MaxGel Extracellular Matrix (ECM) solution</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">E0282</td>
			<td style="width:232px;">See step 11 of the protocol</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Neurobasal</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">21103-049</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:141px;">Penicillin/Streptomycin (Pen/Strep)</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">15140-122</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Retinoic acid (RA)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">R2625</td>
			<td style="width:232px;">Should be stored in the dark at 4&#176; C because this reagent is light sensitive</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">SH-SY5Y Cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:133px;">CRL-2266</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">0.5% Trypsin + EDTA</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">15400-054</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:141px;">Falcon 35mm TC dishes</td>
			<td style="width:141px;">Falcon (A Corning Brand)</td>
			<td style="width:133px;">353001</td>
			<td style="width:232px;">-</td>
		</tr>
	</tbody>
</table>

differentiation of the sh-sy5y human neuroblastoma cell line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

目前，有在神经生物学和neurovirology领域许多情况下，未分化的SH-SY5Y细胞被用作用于人类神经元27-36，和重要的是，未分化的细胞可能缺乏的表型功能模型如最佳的病毒摄取2是必要的准确解释。至关重要的是，使用的SH-SY5Y细胞或任何其他在体外的神经元系统时，将细胞适当地分化成神经元，以获得这是什么可能在神经元在体内发生的最佳可能的表示数据。上述协议的...

Watch this Scientific Journal Video about Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line at JoVE.com

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

differentiation-of-the-sh-sy5y-human-neuroblastoma-cell-line

Research

JoVE Journal

Developmental Biology

70.9K 次观看.  The Pennsylvania State University. It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications. 

视频: Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

在永生多能耳科内皮祖细胞分化启动

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

人成肌细胞的分离，造血分化的评估和存储操作的钙进入测量

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential 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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...