这项工作得到以下资助:CVON（HUSTCARE）:荷兰心血管研究计划（荷兰心脏基金会，荷兰大学医学中心联合会，荷兰卫生研究与发展组织和荷兰皇家科学院）;欧洲研究委员会（ERCAdG 323182 STEMCARDIOVASC）。我们感谢E. Giacomelli（LUMC）帮助hPSC心脏分化。

1.溶液和试剂的制备<ol><li>通过组合表1中所述的试剂，使用低胰岛素，牛血清白蛋白，聚乙烯醇，必需脂质（LI-BPEL）培养基34,35,36制备hPSC-CM培养基。通过0.22μm孔过滤器过滤介质，并在4°C下储存长达2周。 </li><li>通过将1mg人重组纤连蛋白重组在5 mL无菌蒸馏水中，制备人重组纤连蛋白原液，达到200μg/ mL浓度，等分，并储存于-80°C。 </li><li>准备1％（w / v）酶洗涤剂溶液（参见表格材料 ），以帮助从微阵列芯片中去除残留细胞，将1g洗涤剂粉末溶解在温热（约40-45℃）去离子水。让溶液冷却并储存在4°C f或长达一年。 </li></ol> 2. MEA芯片的灭菌（图1A） 注意:可使用多种不同的MEA配置，具有单阱或多阱格式。这里描述的协议使用具有8×8格栅排列的60个记录电极的单室MEA（参见材料表 ）。电极直径为30μm，电极间距离为200μm。还存在一个参考电极。 <ol><li>用去离子水彻底冲洗芯片。 </li><li>将MEA芯片放入可以进行高压灭菌的玻璃培养皿中。将菜包裹在铝箔上。 </li><li>在实验室压力锅中灭菌6分钟。打开前让盘子冷却下来。 注意:或者，芯片可以通过将其浸入1mL 80％（v / v）的乙醇中，在室温下灭菌15-30分钟。 </li><li>将芯片放在细胞培养罩中并暴露表面到紫外光约30分钟。 </li></ol> 3.涂覆MEA芯片（图1B和1C） <ol><li>将清洁的芯片放在标准的10厘米Ø塑料无菌培养皿中。 </li><li>向培养皿中加入8 mL无菌蒸馏水，形成一个加湿室，放置在培养箱中时可防止芯片上的少量培养液干燥。 </li><li>使用定制的聚四氟乙烯（PTFE）环（ 图1B ），以确保电极阵列所在的芯片中心处的心肌细胞的电镀。 （PTFE环预先储存在80％乙醇中）。在培养罩中，从乙醇中取出环，将其放入无菌培养皿中，不用盖子，并允许环在发动机罩中干燥。 </li><li>使用火焰灭菌的镊子将干环放在一个MEA芯片内（ 图1C ）。 注意:或者使用80％乙醇洗涤镊子，并使其干燥使用组织培养罩之前。 </li><li>解冻一等份的人重组纤连蛋白原液（200μg/ mL，在蒸馏水中），并用Ca 2+和Mg 2+稀释在PBS中，得到40μg/ mL的工作溶液。通过在环内加入50μL的40μg/ mL纤连蛋白来涂覆电极。 注意:使用人重组纤连蛋白的涂层确保了最佳的hPSC-CM附着。然而，可以使用其他类型的包衣蛋白，例如牛纤连蛋白或细胞外基质蛋白混合物。 </li><li>关闭10厘米Ø塑料培养皿的盖子，并小心地将含有MEA芯片的培养皿转移到培养箱中。在37℃下孵育至少1小时，或以4°CO / N孵育。 </li><li>将含有MEA芯片的菜转移到细胞培养罩。在使用MEA芯片之前，使用P200移液器或发动机罩中的真空系统抽吸50μL纤连蛋白，而不会移动PTFE环。使用pla这一步的坚定提示。确保没有固体物体（ 例如，移液管尖端）接触盘的内部，因为这可能会损坏电极。 注意:这将延长MEA芯片的使用寿命。 </li><li>轻轻加入950μLLI -BPEL培养基（ 见表1和材料表 ），确保其均匀分布，并且环不浮动。把菜放回孵化器。 </li></ol> hPSC-CMs解离和电镀（图2） 注意:这里描述的方案使用在开始分化后〜18天使用细胞因子34在单层培养物中分化的hPSC-CM。然而，已被证明适用于任何2D和3D hPSC-CM培养。当在较早或较晚的时间点使用分化培养物时，调整解离酶的孵育时间（参见材料表 ）可能是necessary。以下卷是针对12孔板格式（3.8厘米2 ）的单孔。 <ol><li>从hPSC-CMs培养液中吸出培养基。 </li><li>当在组织培养罩下工作时，加入1-2mL /孔的没有Ca 2+ / Mg 2+的PBS以洗涤培养物。吸出PBS。 </li><li>加入500μL/孔的解离酶。在37°C孵育5分钟。 </li><li>加入1 mL LI-BPEL /孔稀释酶。使用P1000移液管轻轻刮擦，轻轻分离hPSC-CM的单层。将细胞悬浮液收集在15 mL管中。 </li><li>用1 mL LI-BPEL冲洗井，收集剩余的所有细胞和细胞团。 </li><li>加入另外2-3 mL的LI-BPEL，达到5-6 mL的最终体积，并用5 mL移液管轻轻移液3-5次，分离细胞团。 注意:在这一点上解离成单个细胞是不必要的，因为它将影响细胞存活。存在小簇将会确保更高的细胞活力。 </li><li>在室温下以300×g离心细胞3分钟。 </li><li>去除上清液，试图去除大部分剩余液体，但不会移动细胞团块。 </li><li>将细胞沉淀物重悬于250μLLI-BPEL（使用P1000和非常温和的移液）。 </li><li>通过在电极阵列的顶部将细胞悬浮液直接吸取到PTFE环的中心，分配〜50μL的每个MEA的细胞悬浮液（可从12孔板的一个孔中制备多达5个MEA）。 注意:在这个阶段，由于细胞簇的存在，细胞难以计数，每个MEA的细胞总数可以基本上变化，这取决于所用的hPSC-CM来源。电镀时，确保解离细胞的云层覆盖电极区域。 </li><li>在37℃下小心地将MEA转移到培养箱中，并允许细胞附着O / N </li></ol>戒指和中等新鲜（图3） <ol><li>电镀后1天，用无菌镊子在无菌环境下小心地取出环（ 图3A-3B ）。 </li><li>在80％（v / v）乙醇中冲洗环，并将其储存在含有新鲜80％（v / v）乙醇的50mL管中。 </li><li>轻轻从MEA芯片中取出500μL培养基，加入500μL新鲜的LI-BPEL。 </li><li>将MEA转移到37℃的培养箱中。 注意:细胞应在戒除和中等改变后1-7天开始跳动（ 图3C ）。 </li><li>在戒除后1-7天测量hPSC-CM在MEA上的电活动。 </li></ol> 6.检查信号质量（图4） <ol><li>打开计算机并启动与MEA建立的链接的软件套件:TCX-Control，MC_MEA Select和MC_Rack。将TCX-Control中的温度设置为37°C，以便在生理温度下记录测量值。 </li><li>取出含有MEA芯片的盘f从孵化器。打开盖子，取出MEA芯片，将其放在纸巾上吸收残留的水分。 </li><li>小心地用纸巾擦拭板的外部触点，并使用用100％（v / v）乙醇浸湿的棉签清洁它们，以清除任何可能导致信号噪音的残留水或碎屑。 </li><li> 将MEA板转移到加热（37℃）记录头阶段以检测自发活动（ 例如，硬件:参见材料表 ;软件:MC_Rack）。 <ol><li>打开MC_Rack:点击"编辑"→"添加MC_Card"来创建一个新的协议。使用下拉菜单"编辑"将不同的录像机和显示窗口添加到协议中。 注意:建议采样频率至少为10 kHz。我们使用的协议包含一个Longterm显示工具，整个MEA芯片的全面布局，以及一个Spike分选机，捕获药物效应至关重要n实时。 Spike Cutout调谐为20 ms的"Pre Trigger"，"Post Trigger"为800 ms，"Dead Time"为2ms。该协议可以保存为&#39;.rck&#39;文件，并在开始实验之前重新加载。 </li></ol></li><li>通过点击"播放"按钮在播放模式下启动协议。 注意:此时可以重新加载在步骤6.5中保存的协议。在MC_Rack中，单击"文件"→"打开"加载协议（.rck文件扩展名）。 </li><li>如果信号显示清晰可见的R峰和T峰，请等待10-15分钟以得出适应阶段。良好和差质量痕迹的例子如图4所示 。 注意:如果在任何电极中没有检测到T峰，请不要进行实验。通常这是由于hPSC-CM的电活动不良或细胞附着到电极的结果。 </li></ol> 7.起始例调查和记录<ol><li>单击"记录"然后"播放"，并在基线条件下获取10分钟的数据，以确定稳态。注意具有最佳信号的电极，以便稍后可以轻松识别和导出以进行分析。 </li><li>对于药物反应评估，每10分钟加入一定浓度的药物。例如，加入终浓度为1μM的hERG阻断剂E4031。为此，取出100μL培养基，并加入溶解在培养基中的相同体积的10μME4031。 注意:如Cavero及其同事31所述，药物溶解体积的明智选择很重要，因为它可以深刻地改变药物反应曲线。 </li><li>对所有感兴趣的其他药物浓度重复步骤7.2。 </li><li>点击"停止"结束协议结尾处的录音。 </li></ol> 8。MEA清洁再利用<ol><li>实验记录完成后，用P1000移液器轻轻取出培养基。不要碰到盘子的内部，因为这会损坏电极。根据当地的安全规定丢弃。 </li><li>使用洗涤瓶用去离子水冲洗MEA碎片，并重复洗涤步骤3-4x。 注意:在这一点上，电池不必完全与芯片分离。 </li><li>向每个孔中加入1mL（v / v）酶洗涤剂溶液，并在4℃下孵育O / N以允许细胞脱离和细胞去除。 </li><li>一天后，用去离子水彻底冲洗MEA芯片，以去除酶洗涤剂溶液和残留的细胞，并加入1 mL去离子水。清洁MEA芯片可以储存在4℃的去离子水中。 </li></ol> 9.数据导出<ol><li>打开链接到MEA设置的MC_Data工具软件。 </li><li>点击"文件"→"打开MC"D"。 </li><li>单击"工具"→"将MCD转换为ABF" 。 </li><li>选择要输出的最佳记录信号的电极进行分析。选择保存导出文件的目录，然后单击"保存"。导出程序可能需要几分钟，具体取决于导出的电极数量和录制文件的总大小。 </li></ol>数据分析<ol><li>下载并安装电生理数据采集和分析程序（ 如 pClamp）。完成后，启动分析软件（ 例如 Clampfit）。 </li><li> RR间隔计算（图5）。 <ol><li>放置两个垂直光标以定义跟踪上的感兴趣区域。选择"事件检测"→"阈值搜索"。水平光标应该交叉所有必须量化的事件， 如图5A所示 。 </li><li>点击＆＃39;确定&#39;，然后"接受整个类别"。然后，软件将搜索所有适当事件的跟踪。蓝点将被放置在事件之上。 </li><li>通过调整主事件检测窗口中的参数来调整自动选择的灵敏度。 </li><li>一旦所有事件都被自动识别，请转到结果窗口（窗口→结果），并复制包含频率数据的标题为"Intervent Interval"的列（ 图5B ）。 </li></ol></li><li> QT间隔计算（图6-9）: <ol><li>在稳态条件下，将一个光标放在单个FP之前，然后放置在单个FP之后。选择"事件检测"→"创建模板"（ 图6 ） 。 </li><li>检查FP是否正确识别，然后单击"添加"。模板将移动到底部面板。 </li><li>将模板另存为&#39;.atf&#39;文件。以这种方式，已经创建了一个模板跟踪，将在整个记录中由软件进行搜索（ 图7 ）。为每个条件创建一个模板，因为药物效应可能会改变FP的形状。如有必要，请稍微过滤轨迹，以在两个光标中最多包含10.000个点。 </li><li>一旦使用&#39;.atf&#39;扩展名保存模板，选择"事件检测"→"模板搜索"并加载模板。 </li><li>调整"模板匹配阈值"，以正确识别所选间隔内的所有FP。 </li><li>一旦所有事件都被正确识别，将它们保存在一个新的&#39;.abf&#39;文件中。 </li></ol></li><li>用分析软件打开文件，并自动计算Q / R和T峰的"峰值时间"（ 图8,9 ）。如果痕迹非常嘈杂，则应用过滤器。通过减去QT间隔计算选择的电子表格编辑器中的T值的Q / R值。 </li></ol>

CLM是Pluriomics bv的联合创始人和顾问。多渠道系统涵盖了出版成本的一部分。

该协议显示了如何使用MEA分离和准备hPSC-CM来测量其FP。 hPSC-CM通常显示自发电活动，其可以作为FP测量，并且可以提供关于跳动频率，QT间期持续时间和心律失常事件的有意义的数据。 需要解离2D心脏分化培养物以在MEA上重新创建跳动层，并且它代表关键步骤。通过重复移液和/或侵蚀性解离酶处理的机械应力可能导致高细胞死亡率，不能附着于MEA平板，以及缺乏自发的电活动。该协议已针对单层培养进行了优化。然而，类似的方法可以用于具有较小修饰的三维（3D）培养物（ 例如，胚状体或EB），例如EB的收集，然后进行PBS洗涤，并且与解离酶的孵育时间更长。进口在2D和3D分化培养物中，分化细胞越老，所需的更长的孵育时间可能是由于细胞外基质沉积增加而分离细胞。 这里描述的用于量化FP参数的方案可以用于产生心脏药物的剂量 - 反应曲线。如Cavero 等人最近描述的31 ，药物的起始浓度可能会深刻影响MEA测量的结果。因此，为了提高结果的准确性和可靠性，我们建议如下:1）在不可逆活化剂/阻断剂的情况下，使用相对较大体积的含有待测试药物的培养基。更详细地，从MEA芯片中除去10-50％的中等体积，并加入等体积的药物先前以适当浓度溶解的培养基。在这种情况下，为了计算最终的药物浓度，至关重要的是考虑呃介质去除后的浓度变化。 2）在可逆活化剂/阻断剂的情况下，从100X储备溶液中加入每种药物剂量10μL。 大多数心脏分化方案导致结节样，心房样和心室样心肌细胞的可变混合群体，心室类型是最具代表性的。当模拟影响特定心肌细胞亚型的心脏疾病或作用于心脏亚型特异性离子通道的药物时，这可能构成限制。虽然几项研究已经优化了条件，以便在心脏分化3,5,37,38中引导更多受控的规范，但其广泛适用性仍在调查之中。 此外，分化的可变效率（在不同的实验和di不同的hPSC线）可能被观察到39,40,41,42,43,44。基于表面蛋白表达35,45（通过荧光辅助细胞分选或通过磁珠选择46,47 ）和代谢选择44,48的心肌细胞富集策略可以表示可应用于任何（转基因或未修改）hPSC线先前电镀hPSC-CM，以改善电信号。 虽然与人成人心肌细胞相比，hPSC-CMs是非常不成熟的，但它们已被证明在r发展和鉴定特定的疾病相关变化（ 例如 ，在通道病变中）19,20,50和药物诱导的反应（ 例如心脏离子通道阻断剂）4,51。此外，未成熟细胞更容易解离，并且在解离和电镀后比成体心肌细胞恢复得更好44因此，hPSC-CM不成熟可能在这方面作为优势得到回报。然而，为了能够总结例如 。迟发性心脏病，忠实地复制成人心肌细胞的药物反应，应该获得更成熟的机械，代谢和电气hPSC-CM状态。成熟这些细胞的方法包括培养时间延长52 ，机械应变53 ，起搏54 ，加小分子55，3D培养物56 ，与其他细胞类型57共培养，甚至这些方法的组合58 ;到目前为止，这些方法都没有导致成人样表型。 作为不成熟特征的一部分，hPSC-CM显示电气自动化。这里提供了如何准确量化QT和RR间隔的细节。测量自发电活动的一个限制是，当hPSC-CM显示不同的跳动频率时，QT间隔的比较可能是困难的。在这种情况下，Bazett或Fridericia的公式可用于校正频率的QT间隔。然而，如前所述， 30 ，我们强烈建议通过绘制原始和校正数据的QT间隔对RR间隔进行主轴回归分析，以排除由于校正方法本身引起的任何可能的偏差。这里提出的方案连同以前描述的方法59,66有助于hPSC-CM FP的程序和分析的标准化，提高数据再现性并允许更好地比较实验室间结果。

人类多能干细胞（hPSC）具有自我更新的能力，并通过分化1,2产生几乎任何细胞类型的人体。已经描述了关于如何将hPSC分化为几个心脏谱系（心室，心房，起搏器样心肌细胞）的详细方案，已经描述了3,4,5,6,7。心肌细胞是电活性细胞，并且他们的电生理活动的详细知识可以非常有助于了解心脏发育和疾病8 。患者特异性hiPSC来源的心肌细胞（hiPSC-CMs）已成功用于建模和研究几种心律失常的细胞，分子和电学特征，包括长QT综合征（LQTS）9,10,11,12,13，Brugada综合征14以及导管蛋白多能性室性心动过速15,16 。此外，已经将多种药物添加到患病的hiPSC-CM中以重述治疗干预并挽救细胞病理表型10,15,20,21,22。最近，已经开发了基于WT hiPSC-CMs的筛选平台，以响应于人类系统对药物发现早期阶段的需求23,24,25 ，因为啮齿动物心肌细胞与hu不同人类离子通道表达和生物物理学26 。 为此，正在开发和实施适用于中高通量应用的技术。这些包括膜电位的光学记录，Ca 2+瞬变和应变，阻抗测量（作为细胞收缩力的间接测量）和细胞外场电位（FP）测量（参见参考文献24 ）。多电极阵列（MEA）器件允许记录由单层或小簇心肌细胞产生和成形的电波形信号（或FP）。 FP轮廓与心脏动作电位相关，并且在一定程度上与心电图（ECG）记录27相关 ;它们通常显示对应于Na +流入和膜去极化（R / Q峰）的初始快速上冲，可能对应于Ca的慢波/平台期2+流出，以及对应于主要K +流出（T峰）的复极相。 FP波形的扰动可以与特定动作电位相位28的变化相关。 虽然动作电位的膜片钳记录可能更有说服力，特别是对于上行速度和静息膜电位的参数，手动测量对于中高通量规则的实验是不可行的，而自动膜片钳最近才被应用于hPSC -CMs 29 。然而，由于MEA上的长时间记录允许研究化合物的急性和慢性暴露，现在可以使用hPSC-CM平台进行药物筛选，发现24,30和安全性药理学31,32 。这是未来精准或者perso的承诺nalized医学33 。 该协议的目的是提供在MEA芯片上分离和电镀hPSC-CM并测量其FP的必要信息。在这个过程中，每个步骤都进行了优化，确保解离后的最佳细胞存活和恢复，最佳细胞与MEA平板的连接以及参数的标准化分析和定量。特别地，解释和例示了细胞外FP记录的过程，QT和RR间期的分析以及药物作用的评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biological safety cabinet/laminar flow hood</td>
			<td>Cleanair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEA2100 in vitro recording system</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 cell culture incubator</td>
			<td>Sanyo</td>
			<td>MCO-15A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hitachi</td>
			<td>himac-CT6EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>MS5</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld pipetman (P-10 (10&#956;L), P-200 (200&#956;L), P-1000 (1000&#956;L))</td>
			<td>Gilson International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips (10 &#956;L, 200&#956;L, 1000&#956;L)</td>
			<td>Corning</td>
			<td>4807 (10 &#956;L), 4810 (200&#956;L), 4809 (1000&#956;L)</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable bottle top filter (0.22 &#956;m pore size)</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic pipette</td>
			<td>Greiner Bio-One</td>
			<td>606180 (5 mL), 607180 (10 mL), 760180 (25 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable Petri dishes</td>
			<td>VWR/ Duran Group</td>
			<td>391-0860</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA chip</td>
			<td>Multi Channel Systems</td>
			<td>MEA200/30iR-Ti-gr</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-169</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant fibronectin</td>
			<td>Tebu-Bio</td>
			<td>J64560</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom made polytetrafluoroethylene (PTFE) MEA rings</td>
			<td>LUMC: department of Instrument Development</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation enzyme - TrypLE Select 1X</td>
			<td>Thermo Fisher Scientific</td>
			<td>12563-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Tergazyme enzyme detergent</td>
			<td>Sigma-Aldrich</td>
			<td>Z273287-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>15230-089</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Reagents for LI-BPEL medium</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21056-023</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>31765-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Free Hybridoma Medium-II (PFHMII)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12040-077</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Bovogen Biologicals Australia</td>
			<td>BSAS05</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate (CDLC)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11905-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) 100X</td>
			<td>Thermo Fisher Scientific</td>
			<td>51599-056</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Monothioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Software version</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>MC_Rack</td>
			<td>Multi Channel Systems</td>
			<td>4.6.2</td>
			<td>Alternatively, data can be recorded using Cardio2D or MC_Experimenter (MultiChannel Systems)</td>
		</tr>
		<tr>
			<td>TCX Control</td>
			<td>Multi Channel Systems</td>
			<td>1.3.4</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA Select</td>
			<td>Multi Channel Systems</td>
			<td>1.3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MC_Data Tool</td>
			<td>Multi Channel Systems</td>
			<td>2.6.15</td>
			<td>Alternatively, Multi Channel Data Manager (MultiChannel Systems) can be used when custom data export is required (HDF5, EDF, etc.)</td>
		</tr>
		<tr>
			<td>Clampfit</td>
			<td>Molecular Devices</td>
			<td>7.0.0</td>
			<td>Used in step 10.1 for analyzing, graphing, and formatting of all of data. To use Clampfit, download and install the electrophysiology data acquisition and pClam (latest version, 10.7.0), available on the Molecular Devices Website. Once complete, launch the software Clampfit. Alternatively, data can be analysed using Cardio2D (MultiChannel Systems) or MatLab custom code.</td>
		</tr>
	</tbody>
</table>

electrophysiological analysis of human pluripotent stem cell-derived cardiomyocytes (hpsc-cms) using multi-electrode arrays (meas)

现在可以从人胚胎和人诱导的多能干细胞（hPSC）中高效地衍生心肌细胞。 hPSC衍生的心肌细胞（hPSC-CM）越来越被认为对人类心血管疾病，特别是心律失常综合征的建模有很大的价值。他们还表现出与体外系统相关的预测药物反应的相关性，这使得它们可能用于药物筛选和发现，安全药理学，或者最终可能用于个性化医学。这将通过从患者或易感个体中获得hPSC-CMs作为hiPSC来促进。然而，对于所有应用，hPSC-CM电性质的精确测量和分析对于识别由于心脏离子通道突变引起的变化和/或靶向离子通道的药物并且可能导致心脏猝死的重要性。与手动膜片钳相比，多电极阵列（MEA）器件具有优势允许中到高通量录音。该协议描述了如何将hPSC-CM的2D细胞培养物分解成小的聚集体和单个细胞，并将它们平铺在多环境协议上以记录其作为场电位的自发电活动。用于分析记录数据以提取诸如QT和RR间隔之类的特定参数的方法也在这里描述。预期这些参数的变化将在携带突变导致心律失常和加入特定药物的hPSC-CM中发生，从而检测那些携带心脏毒性危险的药物。

解离和电镀后的一天，hPSC-CM层将被看作是覆盖MEA室中心的致密和白色膜（ 图3A ）。去除环后（ 图3B ）， &#160;该层应保持原位，光学显微镜上的检查将显示（收缩）hPSC-CM层覆盖的MEA电极（ 图3C ）。由于电池的物理和电耦合，仅使用一个电极（金电极）进行分析。 或者，当使用3D结构（例如胚状体或微组织）时，可以对它们进行电镀，使得它们在物理上和电气上脱离。在显微镜下进行视觉检查可以证实，在MEA之间没有物理连接和非同步的R波，确认没有电耦合。在这c可以分析多个独立的电极。 FP迹线的典型记录如图4所示 。特别地，可以通过存在对应于Na +流入和膜去极化（R / Q峰），对应于K +流出（T峰）的清楚的复极相和高的信噪比（ 图4A ，左:注Y轴刻度和图4B ）。不良质量痕迹（ 图4A ，中间）可能是hPSC-CMs连接到MEA板或弱hPSC-CM电活动的失败的结果。等待1-3天才能更好的贴附可能会提高信号;然而，如果没有可见的信号改善，推荐从实验中排除该MEA。过滤后可以分析嘈杂的痕迹（ 图4A ，右）。 图5A，5B ）。在由垂直光标定义的时间间隔内，应存在与每个峰对应的蓝色标记。如果程序未识别一个或多个峰值，请尝试移动水平光标并再次运行分析，或调整检测设置。类似地，可以通过目视检查显示FP检测的屏幕来识别QT间期的成功分析（ 图7 ）。在由垂直光标定义的时间间隔内，应存在与每个FP检测对应的蓝色标记。如果程序未识别一个或多个FP，请尝试重新定义FP模板（ 图6 ）或调整检测设置并再次运行分析。 提取单个FP踪迹或其平均值（ Figure 8）可以用于获得具有特定设置的QT间隔值， 如图9所示 。 QT-RR关系的分析是可取的，在患病和WT hPSC系中是有意义的（ 图10A ），并评估QT间期校正的需要和/或效果（ 图10B-10D ）。与WT对照相比，携带LQTS引起突变的hPSC-CM具有延长的QT间期（ 图11A ）。用hERG阻滞剂治疗hPSC-CM导致QT间期延长（ 图11B ）;相反，用hERG激活剂治疗可以缩短QT间期（ 图11C ）。最后，影响hPSC-CM的跳动频率的药物治疗应作为RR间期的变化（ 图11D ，RR间期缩短）是可见的。 <img alt="图1" src="/files/ftp_upload/55587/55587fig1.jpg"<html> /&gt; 图1:MEA芯片的灭菌和涂层。 （ A ）代表消毒过程的方案，包括将MEA芯片放置在可高压灭菌的玻璃培养皿中，包裹在铝箔中，蒸煮6分钟，并暴露于UV下30分钟。 （ B ）定制PTFE环的顶部（左面板）和侧面（中间面板）该环具有1.2cm的外径，包括4个翼片，其允许其定位在MEA芯片的中心并且内径为0.4cm（右图）。 （ C ）代表MEA芯片的制备的示意图，包括将芯片放置在标准塑料培养皿中，在MEA室外加入8mL去离子水，将PTFE环放置在室的中间，并用纤连蛋白涂覆电极阵列。孵育后，除去纤连蛋白，用培养基置换。t ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55587/55587fig2.jpg" /> 图2:hPSC-CM的分离和电镀。代表hPSC-CMs酶解离，离心，再悬浮和电镀在MEA室中心的过程的示意图。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55587/55587fig3.jpg" /> 图3:具有hPSC-CM层的MEA芯片。 （ A ）包含环和hPSC-CM层的MEA芯片的顶视图。 （ B ）拆卸后MEA芯片的侧视图。 （ C ）hPSC-CM层的亮场图像，电极阵列4倍放大。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55587/55587fig4.jpg" /> 图4:MEA记录hPSC-CMs。 （ A ）记录有MEA的代表性痕迹显示出良好的质量曲线，R / Q和T峰清晰可见，信噪比高（左），质量不良的痕迹，没有清晰可见的R / Q和T峰（中），和R / Q和T峰的嘈杂迹线清晰可见，但信噪比低（右）。 （ B ）具有不同形态的良好质量的FP迹线的代表性例子，可以使用hPSC-CM进行MEA实验记录。阴影区域表示分析期间测量的QT间隔。由于MEA的FP类似于第一个衍生e的动作电位28 ，我们计算了FP轨迹的积分，如红色虚线所示，作为接近完全动作电位复极化的T波选择的理论演示。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55587/55587fig5.jpg" /> 图5:RR间隔计算。 （ A ）使用分析软件进行自动峰检测（上）和数据提取（底部）的RR间期分析示例（参见材料表）。垂直光标标识感兴趣的时间间隔，水平光标横穿所有由蓝色标记检测和识别的事件。 （ B ）放大列显示用于计算RR间隔的提取数据。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55587/55587fig6.jpg" /> 图6:创建FP模板。使用放置在单个FP之前和之后的垂直光标的模板选择示例。此模板用于FP自动识别。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" src="/files/ftp_upload/55587/55587fig7.jpg" /> 图7:FP模板自动识别。在两个垂直光标定义的间隔期间进行模板搜索的示例。所有检测到的事件都由蓝色标记，并且是自动的y叠加在插图中。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图8" src="/files/ftp_upload/55587/55587fig8.jpg" /> 图8:FP参数的量化。分析保存的事件，手动识别前两个游标中的R峰值和在最后两个游标中手动识别的T峰。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig8large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图9" src="/files/ftp_upload/55587/55587fig9.jpg" /> 图9:分析窗口。统计窗口中使用的参数来检测R峰。对于T峰的检测，更改光标和（如有必要）polarity。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig9large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图10" src="/files/ftp_upload/55587/55587fig10.jpg" /> 图10:QT-RR关系。 （ A ）WT（LQT1 corr ）与Long QT综合征1型（LQT1 R190Q ）hPSC-CM之间的QT与RR间期的不同关系的例子。阴影区域表明，RR间期同样的变化在LQT1病变线的QT间期产生更大的变化，因此可能增加心律失常易感性。 （ B ）来自7个不同hPSC系的CM在MEA测量的未校正的QT和RR间期之间的关系。 QT校正对Bazett（ C ）或Fridericia（ D ）公式的影响显示和可见，因为QT-RR的斜率变化<html> terval关系。参考文献30 。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig10large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图11" src="/files/ftp_upload/55587/55587fig11.jpg" /> 图11:疾病或药物诱导的QT和RR间期变异。 （ A ）与其同基因WT对照相比，源自携带Long-QT综合征（LQTS）突变的患者的hPSC-CM中QT间期延长的实例。 （ B ）hPSC-CM药物hERG阻滞后QT间期延长的实例。 （ C ）用增加剂量的hERG激活剂治疗时QT间期缩短。箭头表示缩短的方向。 （ D ）药物诱发的RR间期缩短实例。参考文献（C）&gt; 30。 <a href="http://ecsource.jove.com/files/ftp_upload/55587/55587fig11large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4"> 低胰岛素，BSA，聚乙烯醇，必需脂质（LI-BPEL）培养基 </td></tr><tr><td colspan="2"> 零件 </td><td colspan="2"> 数量为100 mL </td></tr><tr><td colspan="2"> IMDM </td><td colspan="2"> 43 mL </td></tr><tr><td colspan="2"> F12 </td><td colspan="2"> 43 mL </td></tr><tr><td colspan="2">抗坏血酸2-磷酸（5毫克/毫升蒸馏水） </td><td colspan="2"> 1 mL </td></tr><tr><td colspan="2">细胞培养补充剂（直接代替L-谷氨酰胺） </td><td colspan="2"> 1 mL </td></tr><tr><td colspan="2">青霉素/链霉素</td><td colspan="2"> 0.5毫升</td></tr><tr><td colspan="2">酚红</td><td colspan="2"> 1毫克</td></tr><tr><td colspan="2">无蛋白质杂交瘤中型II（PFHMII） </td><td colspan="2"> 5 mL </td></tr><tr><td colspan="2"> BSA（IMDM中10％wt / vol） </td><td colspan="2"> 2.5 mL </td></tr><tr><td colspan="2"> PVA（蒸馏水中5％wt / vol） </td><td colspan="2"> 2.5 mL </td></tr><tr><td colspan="2">化学定义的脂质浓缩物（CDLC） </td><td colspan="2"> 1 mL </td></tr><tr><td colspan="2">胰岛素 - 转铁蛋白 - 硒 - 乙醇胺（ITS-X）100X </td><td colspan="2"> 0.1 mL </td></tr><tr><td colspan="2"> α-一硫代甘油（13mL，1mL IMDM） </td><td colspan="2"> 0.3mL </td></tr><tr><td colspan="4">将试剂结合，用0.22μm孔过滤器过滤，并将培养基在4℃下储存长达2周。 </td></tr></tbody></table>表1:Li-BPEL培养基组成。

Watch this Scientific Journal Video about Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs at JoVE.com

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

来自人多能干细胞（hPSC-CM）的心肌细胞的电生理学表征对于心脏疾病建模和确定药物反应至关重要。该协议提供必要的信息，用于解离多个电极阵列上的hPSC-CMs并测量其场电位，以及分析QT和RR间期的方法。

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

electrophysiological-analysis-human-pluripotent-stem-cell-derived

Research

JoVE Journal

Developmental Biology

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

19.7K 次观看.  Leiden University Medical Center. 来自人多能干细胞（hPSC-CM）的心肌细胞的电生理学表征对于心脏疾病建模和确定药物反应至关重要。该协议提供必要的信息，用于解离多个电极阵列上的hPSC-CMs并测量其场电位，以及分析QT和RR间期的方法。 

视频: Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

科研

发育生物学

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能干细胞衍生的心肌修复心肌细胞

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...