这项研究部分由Eurostars grant Estars2 113937 CARDIOMYO（EM&DK）和NWO-VICI grant 91818602（JV）资助。

1. 细胞培养基和试剂的制备
<ol><li>通过将 1 mL B27 补充剂 （50x） 添加到 49 mL RPMI1640中来制备 hiPSC-CM 培养基。</li>
	<li>首先将 5 mL 敲除血清替代品加入 45 mL hiPSC-CM 培养基中，制备重镀培养基。然后，加入 22.5 μL Y-27632 ROCK 抑制剂（1：2,000 稀释度;储备浓度：10 mM）并充分混合。</li>
	<li>准备基底膜包被板，如下所述：<ol><li>在冰箱或冰盒中解冻一瓶基底膜过夜。</li>
		<li>用适量的杜尔贝克改良鹰培养基/火腿F12（DMEM/F12）稀释基底膜。由于每批基底膜具有不同的浓度，因此计算达到 1 mg/1 mL 稀释度所需的 DMEM/F12 量。制作0.5或1mL等分试样，在-20°C下储存。 注意：步骤 1.3.2 需要在流罩内的冰上完成。</li>
		<li>解冻基底膜的 0.5 mL 等分试样，并用 6 mL DMEM/F12 稀释。充分混合。</li>
		<li>通过在 24 孔板中移液 250 μL 稀释的基底膜/孔来涂覆板。根据不同孔板格式的每个孔的表面积进行计算，例如，6 孔板中的 1 mL/孔。</li>
		<li>将板在37°C培养箱中孵育1小时。</li>
	</ol></li>
	<li>根据制造商手册，通过将Fura-2 AM溶解在二甲基亚砜（DMSO）中来制备Fura-2 AM等分试样，以制备1 mM等分试样。将等分试样储存在-20°C。</li>
</ol>2. 培养 hiPSC-CMs
<ol><li>在37°C水浴中解冻一瓶hiPSC-CMs。将小瓶的解冻内容物转移到 15 mL 管中。以滴加方式向细胞中加入 10 mL 重镀培养基。</li>
	<li>以100× g离心细胞5分钟。</li>
	<li>除去上清液并加入 1 mL 重镀培养基。轻轻上下移液以重悬沉淀并去除任何细胞团块。</li>
	<li>使用血细胞计数器或自动细胞计数器计数细胞数量。</li>
	<li>从孵育板上取出基底膜并用重镀介质代替。</li>
	<li>将细胞密度适于适当的细胞密度接种在涂有基底膜的 6/12/24/48/96 孔板上（例如，24 孔板中的 250,000 个细胞/孔）。 注意：对于Ca 2 + 瞬态实验，将细胞铺在玻璃底板上。</li>
	<li>第二天用hiPSC-CM培养基替换培养基，随后在24孔板中每隔一天使用0.5 mL /孔更新培养基。</li>
	<li>一旦hiPSC-CMs从解冻和重新铺板中恢复（在本方案中重新铺板后3-5天），执行收缩力测量。 注意：在细胞重镀不均匀的情况下，一组细胞可能不与其余重铺板细胞接触，并可能显示一些不规则或不同步的跳动模式。为避免异步，建议在每个孔中均匀分布细胞和同步的良好连接的单层hiPSC-CM。</li>
</ol>3. 收缩力测量
<ol><li>打开基于光学的测量系统、显微镜发光二极管 （LED） 光源和计算机（参见 材料表）。</li>
	<li>打开程序并打开一个新文件。在屏幕左上角的 “文件” 下，单击“ 新建”。</li>
	<li>单击“收集”，选择“实验”，然后选择“iPSC + 钙”。</li>
	<li>打开气候控制设备。将温度设置为 37 °C，将CO 2 水平设置为 5%。</li>
	<li>用 Tyrode 溶液（24 孔板中的 0.5 mL/孔）替换 hiPSC-CM 培养基。用气候控制盖更换板盖。 注意：如果需要执行钙瞬态测量，请用Fura-2 AM加载电池，如第4节所述。</li>
	<li>一旦气候控制装置显示温度为 37 °C，将板放入基于光学的测量系统内。 注意：收缩是使用明场条件测量的。</li>
	<li>点击 打开单元格查找器 在工具栏下，然后等待弹出新屏幕。在屏幕的右上角，选择 板格式 和 孔。</li>
	<li>选择一个孔，选择 孔内的一个区域以观察同步收缩和松弛，按 开始，单击 添加测量。在设置中选择 测量的持续时间 （每个区域 10 秒）。立即放弃异步收缩测量并选择新区域。 注意：实时收缩瞬变可以在监视器上看到。对于同步收缩，观察到平滑的连续瞬态，而对于异步收缩，观察到小的额外峰值。</li>
	<li>根据井的大小测量井内的多个区域。例如，要遵循这项研究，请在 24 孔板中测量每孔 10 个区域。如果只需要基线测量并且没有计划测试任何化合物，请从步骤3.13继续。</li>
	<li>测量孔内区域后，按 完成 并打开基于光学的测量仪器，将应力源/化合物添加到孔中（例如， 500 nM ISO，溶解在 Tyrode中）。</li>
	<li>根据感兴趣的化合物选择孵育时间（在此协议中使用ISO2分钟）。 </li>
	<li>单击自动测量以允许基于光学的测量仪器在执行基线测量的相同区域执行测量，从而在孔内进行配对 测量 。</li>
	<li>完成孔中的所有测量后，按 完成 。</li>
	<li>从屏幕右上角选择下一个井。</li>
	<li>继续测量所有需要的孔。</li>
	<li>测量完所有必需的孔后，单击 停止 并保存文件。 注意：气候控制用于保持稳定的 CO2。这允许以高速方式测量化合物/应激源对hiPSC-CM的影响。</li>
</ol>4. 钙瞬态测量
<ol><li>准备37°C泰罗德溶液。</li>
	<li>将Fura-2 AM等分试样溶解在Tyrode溶液中，以便将终浓度为1μM Fura-2 AM添加到细胞中。 注意：使用 Fura-2 AM 时，请确保流罩中的灯已关闭，以尽量减少光线暴露。</li>
	<li>吸出培养基并用含有Fura-2 AM的Tyrode溶液代替。</li>
	<li>在培养箱或基于光学的测量系统中在37°C孵育15分钟。</li>
	<li>取出含有Fura-2 AM的Tyrode溶液，然后用新鲜的Tyrode溶液洗涤2x。最后，将细胞在Tyrode溶液中在37°C下再孵育5分钟，以使Fura-2AM脱酯化。</li>
	<li>将板放在基于光学的测量系统中并开始测量，如第 3 节所述。 注意：在步骤 4.6 中。通过与收缩测量同时开始测量，记录比例钙瞬变：在100μm光斑中以340nm和385nm激发;在 510 ± 40 nm 处收集的发射。除了收缩力痕迹外，屏幕上还将出现实时比例钙瞬变。</li>
</ol>5. 数据分析
<ol><li>要分析数据，请单击桌面上的 细胞求解器 。</li>
	<li>单击 导入 并选择要分析的文件。</li>
	<li>程序执行分析后，观察屏幕上出现的接受（蓝色）和拒绝（红色）瞬变（图1A）。 注意：此处使用了以下默认抑制标准，由信噪比（SNR）和拟合优度（R 2）定义：R 2>0.9和SNR >4表示收缩瞬变，R2>0.5和SNR >3表示钙瞬变。用户可以根据测量质量、细胞质量和研究人员的主要结果来调整这些排斥标准。例如，如果研究人员的兴趣主要是松弛动力学，则可以降低峰值变量的时间标准。如果获得非常强的钙瞬变，则可能必须提高SNR以排除噪声较大的数据。</li>
	<li>单击 导出 并勾选平均瞬态数据。检查电子表格中显示的分析数据。</li>
	<li>关闭计算机和所有其他仪器。</li>
</ol>

实时测量 hiPSC-CMs 中的钙和收缩力参数

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EM是CytoCypher BV的员工。MH是CytoCypher BV的首席执行官。其余作者声明，该研究是在没有任何商业或财务关系的情况下进行的，这些关系可以解释为潜在的利益冲突。

在这项研究中，描述了一种对hiPSC-CMs进行收缩力和钙瞬时测量的方法，并研究应激源/药物对这些细胞的影响。收缩性测量，比例钙测量和对ISO的反应在三轮不同的分化中进行，作为生物学重复。对于每个生物重复，在三个孔上进行测量作为技术重复。以这种方式进行测量的原因是为了确保可以评估样品和平台的生物变异性和技术变异性。除了考虑适当数量的生物学和技术重复外，hiPSC-CM培养条件对于获得持续和可靠的结果可以发挥重要作用。在测量过程中，必须符合标准，例如hiPSC-CMs的年龄、培养基的组成、重铺条件和化合物孵育时间。在这项研究中，在一个井中两次测量11个区域，每次10秒，平均需要568±24秒。这包括 79 ± 11 秒的 ISO 孵育时间。这归结为每孔大约 10 分钟，这应该使研究人员能够在 ~4 小时内测量完整的 24 孔板。气候控制用于保持稳定的CO2。这允许以高速方式测量化合物/应激源对hiPSC-CM的影响。
为了测量iPSC-CMs的收缩功能，有几种现有的系统依赖于光遗传学13或无标记视频显微镜5,6。光遗传学系统非常复杂，需要特殊技术才能获得电物理和/或收缩力数据。其他系统依赖于视频的事后分析，这需要大量的存储空间，并且在视频采集过程中缺乏直接反馈。此外，很难实现足够的时间和空间分辨率，这可能会导致欠采样。同样，带有荧光报告基因7的CRISPR / CAS-9编辑的hiPSC-CMs可能会对心肌细胞行为产生不良影响。使用荧光染料进行收缩-弛豫测量也是一种优雅的方法，但限制了对同一样品进行较长时间的实验14。
然而，这种基于光学的测量平台可用于测量收缩-弛豫时间，而无需使用任何荧光染料或侵入性方法。然而，由于像素相关性不能提供空间信息，该方法不能用于测量收缩强度，而只能可靠地检测收缩和松弛速度的变化。引入的测量系统是温度控制的，可以以每秒>200帧的分辨率实时获取钙和收缩力数据。此外，每个测量的位置都被存储，这使得配对测量成为可能，从而提高实验的能力。此外，该平台为研究人员提供了存储数据并在实验后立即分析数据的能力。总体而言，这种基于光学的测量系统被证明是研究细胞病理机制的一种有前途的方法，可以评估化合物对hiPSC-CMs功能的影响，并且有助于药物筛选的临床前过程。

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65326">Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes</a>. The Authors section was updated from:
Rafeeh Dinani1,2 
Emmy Manders1,2,3 
Michiel Helmes1,2,3 
Lili Wang4 
Bjorn Knollmann4 
Diederik W. D. Kuster1,2 
Jolanda van der Velden1,2 
1Division of Physiology, Amsterdam UMC, Vrije University 
2Heart Failure &#38; Arrhythmias, Amsterdam Cardiovascular Sciences 
3CytoCypher BV 
4Division of Clinical Pharmacology, Vanderbilt School of Medicine
to:
Rafeeh Dinani1,2 
Emmy Manders1,2,3 
Michiel Helmes1,2,3 
Lili Wang4 
Bjorn C. Knollmann4 
Diederik W. D. Kuster1,2 
Jolanda van der Velden1,2 
1Division of Physiology, Amsterdam UMC, Vrije University 
2Heart Failure &#38; Arrhythmias, Amsterdam Cardiovascular Sciences 
3CytoCypher BV 
4Division of Clinical Pharmacology, Vanderbilt School of Medicine

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65326">Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes</a>. The Authors section was updated.

Erratum: Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

心力衰竭是全世界死亡的主要原因。2019年，心血管疾病在全球造成1860万人死亡，比过去十年增加了17.1%1。尽管研究人员努力确定预防和治疗心力衰竭的药物靶点，但患者的预后仍然很差2。动物模型相对于人类的病理生理学差异可能是心力衰竭治疗优化成功有限的潜在因素之一3。需要额外的类人模型来模拟疾病并测试新型药物化合物的毒性和有效性。
人诱导的多能干细胞来源心肌细胞（hiPSC-CMs）是确定应激源、缺血、代谢改变或致病基因变异诱导的细胞病理机制以及药物干预的有效性和毒性的有力工具4。为了确定对hiPSC-CMs功能特性的影响，需要一种高速系统，以无偏和可重复的方式测量细胞收缩的收缩和舒张特性。当前研究的总体目标是证明基于光学的系统是对hiPSC-CMs的功能参数进行实时分析的强大工具。
目前，有多个平台可以评估hiPSC-CMs的收缩性。然而，目前的系统要么读数慢，要么所需的细胞数量可能是一个挑战。无标签视频测量系统5、6 依赖于对视频的事后分析，这需要大量的存储空间，并且在视频采集过程中缺乏直接反馈。此外，很难实现足够的时间和空间分辨率，并可能导致欠采样。确定心肌细胞特性的其他方法，例如带有荧光报告基因的成簇规则间隔短回文重复序列（CRISPR）/ Cas9编辑的hiPSC-CMs7 可能会干扰细胞的基因稳定性，并且需要专门的实验室专业知识。
为了克服上述限制，本研究开发并引入了一种独特的基于光学的测量系统。该平台只需在任何所需的板格式上重新镀层，即可对未修饰的hiPSC-CM进行收缩性测量，而不受板尺寸限制。此外，实时测量允许直接观察和分析hiPSC-CMs的功能参数，从而提供即时调整和优化方案的实验设置。此外，每个测量的位置都被存储，这使得可以对同一样品进行配对测量，从而提高实验的能力。
为了演示基于光学的系统，在对照hiPSC-CM生产线上进行了收缩性测量。该对照hiPSC系由具有正常核型8的健康男性供体的真皮成纤维细胞产生。在37 °C下测量收缩动力学，基于在250 Hz采样频率下弛豫期间相对于参考系的像素相关性变化。由于收缩的确切开始并不总是明确确定的，因此将达到峰值高度的20%的时间点（到峰值的时间20%）作为测量峰值时间的起点。通过这样做，在一个样本中发现该参数的变异性较小。同样，由于难以评估信号返回基线的确切时刻，因此使用从峰值达到基线返回基线的80%（到基线的时间80%）来描述松弛时间。
总体收缩性测量包括静息跳动频率，从20%峰值到峰值收缩的时间（TP.Con），以及从峰值收缩到基线80%的时间（TB.骗局80）（图1B）。为了测试应激源的效果，将细胞与异丙肾上腺素（ISO）一起孵育。此外，与收缩性测量巧合的是，通过在250 Hz的采样频率下加载Fura-2-乙酰氧基甲酯（Fura-2 AM）来测量Ca2 +瞬变。 使用超开关的比例Ca2+测量9在直径为50μm的照明区域上进行，对应于收缩性测量的区域。Ca 2+测量数据表示为Ca2+瞬态（TP.Ca）中从20%峰到峰的时间，以及从峰值到基线80%的时间（TB.Ca80）（图1C）。使用快速、自动的数据分析工具来生成每个区域的平均收缩和钙动力学参数。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>B-27 Supplement (50x)</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
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			<td>CytoSolver transient analysis tool</td>
			<td>CytoCypher</td>
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			<td>A fast automatic data analysis tool</td>
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			<td>DMEM/F-12</td>
			<td>Thermo Fisher</td>
			<td>11320033</td>
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			<td>Fura-2, AM</td>
			<td>Thermo Fisher</td>
			<td>F1221</td>
			<td></td>
		</tr>
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			<td>Isoprenaline hydrochloride</td>
			<td>Merck</td>
			<td>15627</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut&#8482; Serum Replacement</td>
			<td>Thermo Fisher</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Merck</td>
			<td>CLS3542C</td>
			<td>Basement membrane</td>
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			<td>MultiCell CytoCypher with Nikon 20x Super Fluor objective and 730 nm LED light source and Ionwizard software</td>
			<td>CytoCypher</td>
			<td></td>
			<td>Optics-based measurement system</td>
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			<td>ROCK inhibitor Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
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			<td>RPMI 1640</td>
			<td>Thermo Fisher</td>
			<td>11875119</td>
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			<td>Tyrode</td>
			<td></td>
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			<td>Self-made solution with final concentration of the following components: NaCl 134 mM, KCl 5 mM, Hepes 12 mM, MgSO4 1.2 mM, NaH2PO4 H2O 1.2 mM, Glucose 11 mM, Sodium Pyrovate 5 mM, 1 mM CaCl2</td>
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real-time measurements of calcium and contractility parameters in human induced pluripotent stem cell-derived cardiomyocytes

人诱导的多能干细胞衍生心肌细胞（hiPSC-CMs）是研究突变介导的心肌细胞功能变化以及确定应激源和药物干预效果的有力工具。在这项研究中，证明了这种基于光学的系统是评估2D中hiPSC-CMs功能参数的有力工具。通过使用该平台，可以在保存完好的温度环境中对不同的板布局进行配对测量。此外，该系统为研究人员提供了即时数据分析。
本文描述了一种测量未修饰的hiPSC-CMs收缩力的方法。在37°C下测量收缩动力学，基于相对于在250 Hz采样频率下弛豫时拍摄的参考帧的像素相关性变化。此外，可以通过用钙敏感荧光团（如Fura-2）加载细胞来同时测量细胞内钙瞬变。使用超开关，可以在直径为50μm的照明点上进行比例钙测量，对应于收缩性测量的面积。

按照该协议，进行了收缩性，Ca2 + 瞬变和对hiPSC-CMs中化合物的反应的测量。通过使用这种基于光学的测量系统，可以简单地将hiPSC-CMs重新镀在所需的板上，并且可以进行收缩性测量。通过计算相对于参考帧的像素相关性，在 100 μm x 100 μm 感兴趣区域 （ROI） 中以每秒 250 帧的速度测量收缩性。系统在舒张末期自动检测参考系。在收缩测量的同时，记录比例钙瞬变。
在这项研究中，使用三轮不同的分化作为三个生物学重复。对于每批分化，测量三口井作为技术重复。这项研究是使用24孔板进行的，并测量了每个孔内的10个区域。测量后，使用CytoSolver程序立即分析数据。报告了以下参数：静息跳动频率，TP。骗局和结核病。对照hiPSC-CMs的Con80（图2A-C）。为了可视化每个上述参数的数据变异性，每个孔内的测量值和每轮分化的三个孔的平均值表示为超图10。为了更好地评估每轮微分和三轮数据的变化，计算了每个参数的方差系数（图2D）。如图2D所示，对应于一个生物重复的技术重复值彼此落在非常接近的范围内（即低方差系数），这说明了该方法的稳健性。
此应用程序非常适合测试药物效果。在这里，测试了500nM ISO（一种非选择性β-肾上腺素能受体激动剂）对对照hiPSC-CMs收缩力的影响。已知这种化合物可以增加心率并发挥积极的促甲状腺作用.如图3A所示，ISO显著提高了所有孔的跳动频率。ISO也增加了动力学，这在TP的降低中很明显。骗局和结核病。缺点80（图 3B，C）。总体而言，在用ISO孵育时，所有孔的跳动频率均显着增加，收缩和弛豫时间减少，这表明该平台记录了对该化合物的预期响应。
在进行收缩力测量的同时，还进行了钙测量。与收缩性数据（图2）相同，hiPSC-CMs的钙测量数据如图 4所示。TB.Ca80 比分离的大鼠或小鼠心肌细胞慢得多，这部分是由于物种和心率差异11，以及hiPSC-CMs中由于肌浆网内的钙动力学较低和钙处理蛋白的表达低于原代细胞而导致的未成熟钙处理12.与收缩性数据类似，计算了钙测量的方差系数，并且在技术和生物学重复中显示出低变异性（图4C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65326/65326fig01.jpg"> 图 1：实验后立即使用 CytoSolver 瞬态分析工具分析数据。 （A）在井内测量的一个区域的可接受瞬态（全部为蓝色）的示例。图A1描绘了收缩瞬变。图A2代表钙瞬变。（B）在一轮分化的三口井中测量获得的平均收缩瞬态。从每个单个或平均瞬态，数据从不同时间点到峰值（TP.缺点）和到基线的时间（TB.Con）的收缩瞬态可以提取。由于收缩的确切开始并不总是明确确定的，因此以峰值时间20%作为测量峰值时间的起点。同样，信号返回基线的确切时刻也很难评估。因此，到基线80%的时间用于描述松弛时间。在这里，TP。缺点（达到峰值的时间 - 达到峰值的时间 20%）和 TB。使用Con 80（到基线的时间80% - 达到峰值的时间）。（C）从一轮分化的三口井测量中获得的平均钙瞬态数据。从每个单个或平均钙瞬变中，可以提取钙瞬变到峰值（TP.Ca）和到基线时间（TB.Ca）的不同时间点的数据。对于达到峰值的时间测量，起点是达到峰值的时间 20%，对于到基线的时间，终点是到基线的时间 80%。在本研究中，使用了 TP.Ca（达到峰值的时间 - 达到峰值的时间 20%）和 TB.Ca 80（到基线的时间 80% - 达到峰值的时间）。<a href="https://www.jove.com/files/ftp_upload/65326/65326fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65326/65326fig02.jpg"> 图2：对三轮不同的分化进行的基线收缩力测量。 每轮分化三个生物重复和三个孔（即三个技术重复）。每轮分化的三个主要符号代表每口井内测量区域的平均值（褪色背景符号）。数据显示为 SEM ±平均值。 有关静息跳动频率的数据以赫兹表示，TP 的数据以赫兹表示。骗局和结核病。Con80，数据以秒为单位。（一）静息跳动频率。（B） 达到峰值的时间（TP.缺点）。（C） 达到基线的时间 80% （TB.缺点80）。（D）为了评估每轮获得的数据之间的差异以及所有三轮分化之间的差异，使用每口井的平均值计算方差百分比系数。 <a href="https://www.jove.com/files/ftp_upload/65326/65326fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65326/65326fig03.jpg"> 图 3：在基线 （-） 和用 ISO （+） 孵育后的收缩力测量，用于三轮分化。 （A）静息跳动频率（-）和用500nM ISO（+）孵育后的跳动频率。（B） 达到峰值的时间（TP.缺点）在基线和用ISO孵育后的测量。（C） 达到基线的时间 80% （TB.Con80）在基线和用ISO孵育后的测量。为了评估ISO对收缩性参数的影响，进行了双向方差分析与邦弗朗尼校正。数据显示为平均± SEM。 有关静息搏动频率的数据以赫兹和 TP 表示。骗局和结核病。Con80，数据以秒为单位表示。<a href="https://www.jove.com/files/ftp_upload/65326/65326fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65326/65326fig04.jpg"> 图4：三轮分化对三口井进行的基线钙瞬态测量 。 （A）达到峰值的时间（TP.Ca）和（B）到基线的时间80%（TB.Ca80）的钙瞬变。数据显示为 SEM ±平均值，TP.Ca 和TB.Ca 80 个数据以秒为单位表示。（C）为了评估每轮获得的数据之间的差异以及所有三轮分化之间的差异，使用每口井的平均值计算方差百分比系数。 <a href="https://www.jove.com/files/ftp_upload/65326/65326fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

作者聚焦：实时测量人诱导多能干细胞来源的心肌细胞中的钙和收缩力参数  

本文描述了一种使用基于光学的平台在人诱导的多能干细胞衍生心肌细胞中进行收缩性和钙测量的既定方法。该平台使研究人员能够以快速且可重复的方式研究突变的影响以及对各种刺激的反应。

实时测量人诱导多能干细胞来源心肌细胞中的钙和收缩力参数

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...

人类诱导的多能干细胞来源的心肌细胞是研究突变介导的心肌细胞功能变化以及定义应激源和药物干预效果的有力工具。需要一个平台，以用户友好和可重复的方式测量人诱导的多能干细胞来源心肌细胞的收缩力和钙处理。该方法使研究人员能够通过像素相关性研究人诱导的多能干细胞来源的心肌细胞的收缩性，并通过用钙敏感荧光团加载细胞同时测量细胞内钙瞬变。通过使用该平台，可以在保存完好的温度环境和不同的板格式下进行配对测量，并立即访问数据。该方法能够研究细胞病理机制并评估化合物的作用和人iPSC来源的心肌细胞的功能，这可能有助于临床前药物筛选过程。

real-time-measurements-calcium-contractility-parameters-human-induced

Research

JoVE Journal

Biology

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

1.7K 次观看.  Vrije University. 人类诱导的多能干细胞来源的心肌细胞是研究突变介导的心肌细胞功能变化以及定义应激源和药物干预效果的有力工具。需要一个平台，以用户友好和可重复的方式测量人诱导的多能干细胞来源心肌细胞的收缩力和钙处理。该方法使研究人员能够通过像素相关性研究人诱导的多能干细胞来源的心肌细胞的收缩性，并通过用钙敏感荧光团加载细胞同时测量细胞内钙瞬变。?...

视频: 作者聚焦：实时测量人诱导多能干细胞来源的心肌细胞中的钙和收缩力参数  

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

人诱导的多潜能干细胞源性心室心肌细胞亚型特异光学动作电位记录

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.