Funding Sources: The University of Arizona, The Jim Himelic Foundation, and the Arizona Center for the Biology of Complex Diseases.

人iPS细胞是从从4毫米皮肤钻取活组织检查分离并通过仙台病毒介导的重编程11在内部重新编程的人真皮成纤维细胞衍生的。亚利桑那州的机构审查委员会的批准大学为对象招募和活检集合中的所有程序。 1. IPSC的文化细胞外基质表面涂层的制备<ol><li> hiPSC文化上的iMEFs日益合流前一天，准备外基质（基质胶）涂层板。 </li><li>缓慢解冻的细胞外基质的等分试样（批号相关的，按照该规格表的说明）在冰上在4℃2小时。 </li><li>在生物安全罩，使用冷枪头（储存在-20℃），加入1等份的细胞外基质的冷Dulbecco氏改良Eagle培养基/营养混合物F-12（DMEM / F12 / HEPES）根据许多特定稀释因子。 </li><li>免除〜60081;每每个新的细胞培养物井外基质L的处理的12孔板需要的。 </li><li>在室温下孵育该板进行几个小时。在这里，用3小时的培养时间。 注:将板可以立即使用或储存于4℃多达两个星期。 </li></ol> 2.将iPS细胞对IMEF饲养细胞生长到细胞外基质的传播<ol><li>关于传代当天，除去从4℃的矩阵涂板，并允许所述板到适应室温在组织培养罩1小时。 </li><li>吸从1井hiPSC培养物的6孔板上的iMEFs生长培养基并用500μL的胶原酶分解试剂代替（1毫克/毫升在基础培养基）。 </li><li>在37℃，10〜 - 30分钟，直到的iPSC菌落边缘出现小幅提升。 注:孵化时间是线依赖性和将变化。显示器在显微镜下分离。 </li><li>汽车efully吸出解离试剂和用磷酸盐缓冲盐水（PBS，pH7.4）中洗轻轻2次。 </li><li>添加1毫升室温mTeSR1完全培养基与10毫微克/毫升Rho相关的蛋白激酶抑制剂（Y-27632 ROCK抑制剂）到孔中。 注:此初始通路期间使用IMEF空调介质和mTeSR1的50:50混合物，建议并且可以是用于人iPS细胞的这种媒体转换期间的存活益处。 </li><li>使用部分放置在罩内的小相差显微镜，手动得分并挑选1 - 2 hiPSC菌落（〜700 - 1000微米直径）使用注射器和针（25克，1.5英寸）。蹬殖民地的得分件与P200的枪头的媒介。 </li><li>吸并从新盘基体处置，小心不要去打扰涂层。 </li><li>转移1ml得自老井含有得分的iPSC成涂好新的矩阵的细胞悬浮液。 </li><li>解放军在37℃CE在孵化器的板，5％ 的 CO 2。岩石在几个快，背部的往复板和侧到另一侧的运动，以均匀地分布的细胞。请勿打扰板24小时。 </li><li>执行与mTeSR1完全培养基（除了Y-27632 ROCK抑制剂不需要在步骤2.5初始电镀后），每日完整的媒体的变化。 </li></ol>矩阵涂层钢板iPS细胞3.小型传代注意:一般来说，人iPS细胞的初始传送到矩阵包被的板后，细胞应再次以类似的方式传代，以确保所有的iMEFs已被淘汰和细胞已适于馈线 - 自由的条件。 <ol><li> iPSC的汇合前一天准备新涂矩阵板在第1节的讨论。 </li><li>上通道的当天，除去从4℃孵育基质涂层板，并允许所述板到适应室温1小时在组织尽头TURE罩。 </li><li>从吸1井iPS细胞的6孔板（最初是从进纸器矩阵转换）的媒介和500μL细胞分离缓冲液进行更换。 注:此处使用的商业细胞解离缓冲液（见材料表）更适合用相比胶原酶mTeSR1介质使用。 </li><li>在室温下孵育3 - 7分钟，直到的iPSC集落的边缘出现稍微抬起。如前所述，培养时间是线依赖性和可变的。因此，监视在显微镜下的细胞解离，以确定的最佳时间。 </li><li>小心吸出解离缓冲液，并用PBS轻轻洗2次。 </li><li>添加500μL室温mTeSR1完全培养基用10ng / mL的Y-27632 ROCK抑制剂到孔中。 </li><li>代替如步骤2.6的得分菌落，用P200尖端推菌落所需数量到媒体并轻轻磨碎细胞悬液的2倍股份ainst井的一个角落，打破hiPSC殖民地变成〜50团块 - 尺寸200微米。例如，对于一个12孔板接送1 - 2菌落转移到每个1新井。传送从老的细胞悬浮液以及成12孔板的单个新井。 </li><li>摇动平板在几个快，背部的往复，并且边对边动作来均匀地分布所述细胞，并让24小时的板其余在37℃，5％CO 2培养箱。 </li><li>执行与mTeSR1完全培养基每日完整的媒体的变化。如前面提到的，这是没有必要的初始镀覆后添加Y-27632 ROCK抑制剂。 注:来自步骤3.7未使用的iPSC集落可以是在这一点上容易冻存。 </li></ol> 4.制备室载玻片和盖玻片为iPS细胞的免疫细胞化学播种注:以下协议采用4孔塑料玻片，用都是适宜12毫米盖玻片在多井的培养基E卷来支持经济有效的免疫。但是，如果使用较大的井和盖玻片大小的期望能够提高介质的卷。 <ol><li>一天前hiPSC汇合，准备室玻片新涂与细胞外基质的第1节中讨论补充足够的基质溶液（〜300 - 每4井室幻灯片以及500微升），充分大衣每良好，没有任何忧虑蒸发。可替代地，将1清洁和灭菌盖玻片到24孔板的每个孔中，涂层用500微升的细胞外基质，并确保盖玻片不漂浮在溶液的顶部之后。 </li><li>上通道的一天，从4℃除去涂覆室载玻片/盖玻片并允许驯化至室温在组织培养罩1小时。 </li><li>从1井人iPS细胞的12孔板的吸出培养基并用500μL的解离缓冲液替换。 </li><li>孵育在室温坦佩叉涂抹了〜3 - 7分钟，直到的iPSC菌落边缘出现小幅提升。 </li><li>小心吸出解离缓冲液，并用10纳克/毫升Y-27632 ROCK抑制剂添加室温mTeSR1完全培养基的1毫升到孔中。 </li><li>使用放置在组织培养罩内的小相差显微镜，手动选择一个4孔腔式载玻片或1新盖玻片1集落/新井待镀（〜1000 - 1500微米的直径），使用P200的吸管尖通过推它从板表面到培养基中。 </li><li>从新室幻灯片/盖玻片吸矩阵，小心不要去打扰涂层。 </li><li>针对很好地打破了hiPSC殖民地的一个角落2次轻轻磨碎细胞悬液成〜50团块 - 尺寸200微米。转移的细胞悬液250μL的从旧井到4孔腔式载玻片/盖玻片的每个新井。 </li><li>带来每一个新的容量以及高达500μL与mTeSR1续癌宁10毫微克/毫升Y-27632 ROCK抑制剂。 </li><li>放置在孵化室滑动/盖玻片，在37℃，5％ 的 CO 2。 </li><li> 24小时之后，用mTeSR1完全培养基每日完整的媒体的变化。再次如前面提到的，添加Y-27632 ROCK抑制剂的初始镀覆后是没有必要的。 </li></ol> 5.准备为iPS细胞的免疫细胞化学<ol><li>一旦汇合，通过从孔中除去培养基并加入预热小心 4％低聚甲醛保留通过固定的hiPSC菌落（〜300微升/孔的4孔腔式载玻片/盖玻片）。在室温下孵育20分钟。 </li><li>适当地根据机构生物危害和化学安全指引弃多聚甲醛。 </li><li>洗涤细胞3次，每次5分钟，在用PBS台式摇床。 注:在腔室玻片细胞/盖玻片可以在4℃无限期在这个阶段存储有充足的PBS。可替代地，它们可以立即用于IC卡。 </li></ol> 6.免疫染色用多能标记iPS细胞的注意:免疫显iPS细胞具有多能性标准的抗体。作为一个例子，在这里细胞是双染色的抗体针对一种1表面和以顺序方式1的细胞内抗原如下:抗阶段特异性胚胎抗原-4（SSEA4）用抗八聚体结合蛋白4（OCT- 4）和抗TRA-1-60具有抗SRY相关HMG盒基因2（SOX2）（请参阅抗体细节材料表）。 <ol><li>从孔中吸出PBS并在500微升/孔封闭溶液添加不含Triton-X-100。孵育在室温下1小时。 </li><li>准备预先确定的浓度（这里，用1:200）的表面的抗体，无论是SSEA4或TRA-1-60，通过在封闭液不含Triton X-100的稀释。 <ol><li>对于玻片，准备了4孔腔式滑动300微升/孔通过混合封闭液上方和抗SSEA4或TRA-1-60一抗的1.5微升的298.5微升。 </li><li>为盖玻片，通过混合封闭液和抗SSEA4或TRA-1-60一抗的1微升199微升制备40微升/盖玻片。 </li></ol></li><li>吸从玻片封闭液和分配的主要抗体为适当的井。对盖玻片将一块封口膜牢牢培养皿内并分配在上面作为一个液滴的主抗体的40微升。使用弯曲的注射器针头和镊子小心抬起盖玻片出培养的井，并放置电池侧向下到抗体上的封口膜的压降。在4℃孵育过夜。 </li><li>第二天早上，洗细胞3次，5 - 每10分钟，通过从腔室滑动除去抗体并在PBS中加入。对于盖玻片，抬起一次性封口膜，并把它早在24孔板，细胞面朝上的好。小心加入PBS。 </li><li>在封闭液500:1，准备适当的二级抗体。 <ol><li>对于腔室玻片，准备300μL/孔的4孔室载玻片通过混合499微升封闭液和1微升的山羊抗小鼠IgG3的的Alexa Fluor 488（为SSEA4）或1微升的山羊抗小鼠IgM的Alexa Fluor 488（用于TRA-1-60）。 </li><li>为盖玻片，通过混合封闭液499微升制备40微升/盖玻片和1微升的山羊抗小鼠IgG3的的Alexa Fluor 488（为SSEA4）或1山羊μL的抗小鼠IgM的Alexa Fluor 488（为TRA-1-60 ）。 </li></ol></li><li>从室滑动吸出PBS中并分配所述第二抗体为适当的孔中。为盖玻片，下列步骤6.2，翻转的盖玻片上，在室温下放置在封口膜2小时抗体溶液。 注意:如果使用荧光标记的二级抗体，该细胞必须避光保存。放置玻片和盖玻片在黑暗的ENV孵育和洗涤过程中ironment。 </li><li>洗涤细胞3次，每次5 - 10分钟各由从腔室滑动除去抗体并在PBS中加入。为盖玻片，从封口膜小心地每一个和放回一个24孔板，电池侧上的孔中。轻轻洗涤过程中添加PBS。 </li><li>之前探测第二抗原，细胞内的OCT-4或在此情况下SOX2，在阻断用透试剂（的Triton-X-100）在室温下1小时制成溶液再次孵育。 </li><li>免疫染色为OCT-4或SOX2，只要按照在步骤6.1-6.6用于施加第一和第二抗体描述的方法。对于此协议的目的，使用OCT-4和SOX2以1:1的浓度:200和二级抗体的山羊抗兔IgG的Alexa Fluor 594以1:500。 </li><li>上述主要和次要抗体治疗后，洗细胞3次，每次5 - 10分钟各用PBS并用4染液&#39;，6&#39;-二脒基-2--phenylindole，二盐酸盐（DAPI）如果核可视化需要按照标准协议。 </li></ol> 7.准备查看<ol><li>对于腔室玻片，小心地从滑动除去上室，按照制造商的说明进行操作。然后，漂洗在蒸馏水中，添加封固剂和盖玻片启用显微镜。 </li><li>为盖玻片，反转上的安装件中置于玻璃显微镜载玻片一滴盖玻片。 </li><li>标准或共聚焦荧光显微镜下的图像。 </li></ol>

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The authors have nothing to disclose.

这里介绍的系统协议提供了一个节省时间和成本效益的方法，在一个按比例缩小的培养技术的形式，专门通过免疫组化来支持有效的多能性的分析。 是所描述的方法的主要优点如下。需要传统上超过3至4个通道，以过渡从饲养层的iPSC馈线-自由培养条件，以消除使用散装解离技术后剩余的残余的iMEFs和典型单层形态出现12,13。与此相?...

重新编程人成年体细胞到诱导多能干细胞（iPS细胞）提供了一种方法，以获得患者特异性的细胞的潜在无限供给到研究疾病1,2。扼要体外的疾病表型将使得可信检验与疾病相关的细胞和分子机制，增强药物发现和个性化药物3。此外，人iPS细胞（iPS细胞）提供导出其可以用作唯一的资源来替换死亡或功能失调的细胞，并在几个障碍4的范围内，恢复功能的特定细胞类型的可能性。 以使用上述应用程序的iPSC的重要前提是，确保在培养膨胀过程中他们的多能性和分化状态被维持。通常情况下，techniq的UE，如流式细胞术，Western印迹，聚合酶链反应和功能分析，这需要大量的细胞和专用设备，用于IPSC的多能性6，7，8，9，10的详细的分析。然而，iPS细胞"未分化状态的常规评估可能有效地通过这些细胞的有限传播专门为免疫细胞化学（ICC）来实现，因此，涉及减少时间和资源。 最新进展允许的iPSC的所限定的无血清条件下生长的，它是在需要的鼠成纤维细胞饲养层和含血清介质常规培养系统一个显著改善。然而，目前的文献不包括描述如何尽快过渡馈线iPSCs的逐步明确协议层到无饲养系统。 在此背景下，本协议系统详细说明了如何生长在辐照的小鼠胚胎成纤维细胞人iPS细胞（IMEF）饲养层可以是（1），适于在无血清培养基中传播，和（2）在小规模培养的特别支持稳健免疫细胞化学分析。总体而言，这种方法代表了用于传播在无血清条件人类iPS细胞用于确认使用免疫细胞化学在常规基础上它们的多能及时和成本有效的方法。

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			<td>DMEM-F12/HEPES</td>
			<td>Life Technologies</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-Basic</td>
			<td>Cell Sciences</td>
			<td>CRF001B</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td>R&#38;D</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Life Technologies</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Basal Medium</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 5X Supplement</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Buffer</td>
			<td>StemCell Technologies</td>
			<td>07174</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M PO4 Buffer</td>
			<td>In-House</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldeyde, prill</td>
			<td>Electron Microscopy Sciences</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phoshate Buffered Saline</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210072</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-4A (C30A3) Rabbit mAb, 
			Sox2 (D6D9) Rabbit mAb, 
			SSEA4 (MC813) Mouse mAb, 
			TRA-1-60(S) (TRA-1-60(S)) Mouse mAb</td>
			<td>Cell Signaling 
			Cell Signaling 
			Cell Signaling 
			Cell Signaling</td>
			<td>2840 
			3579 
			4755 
			4746</td>
			<td>Alternatively, a combination of 6 pluripotency primary antibodies can be purchased together as a kit in Catalog #9656</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgM Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21042</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgG3 Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21151</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rb IgG Alexa Fluor 594</td>
			<td>Life Technologies</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Cell Culture Plates</td>
			<td>Fisher Scientific&#160;</td>
			<td>0720080/0720081</td>
			<td>Available in 6, 12, 24, 48, 96 well sizes</td>
		</tr>
		<tr>
			<td>Chamber Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 565 21</td>
			<td>Available in Glass or Permanox Plastic in 1, 2, 4, 8, 16 well sizes</td>
		</tr>
		<tr>
			<td>Coverglass for growth</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 545 82</td>
			<td>Available in 12, 15, 18, 22 and 25mm sizes</td>
		</tr>
	</tbody>
</table>

small-scale propagation of human ipscs in serum-free conditions for routine immunocytochemical characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

这个协议提供的iPSCs的人类如何可以从饲养层转移至无饲养条件下，并随后传播以有限的方式来具体实现，用于确认多能性维持成本效益的免疫细胞化学的逐步描述。 图1显示了该协议的示意图。 图2A示出hiPSC菌落在6孔板的iMEFs生长。这些殖民地表现出有确定的边界和密相明亮的中心典型形态。 如图2B所示，在12孔板的iPSC移交后馈线-自...

Watch this Scientific Journal Video about Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization at JoVE.com

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

small-scale-propagation-human-ipscs-serum-free-conditions-for-routine

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  The University of Arizona. Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

视频: Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

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 ...

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 ...

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...