Name: generation of ventricular-like hipsc-derived cardiomyocytes and high-quality cell preparations for calcium handling characterization
Uploaded: 2020-01-17T12:00:00
Description: Watch this Scientific Journal Video about Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization at JoVE.com

这项研究得到了AHA科学家发展赠款17SDG33700093（F.S.） 的支持;西奈山KL2学者奖临床和转化研究职业发展KL2TR001435（F.S.）;NIH R00 HL116645 和 AHA 18TPA34170460 （C.K.）。

在这项研究中，使用成年大鼠心肌细胞的实验是在西奈山伊坎医学院批准的机构动物护理和使用委员会（IACUC）协议下进行的。成年大鼠心肌细胞被分离出斯普拉格道利鼠的心脏，通过兰根多夫的方法，如前16所述。
1. 媒体的准备
<ol><li>准备 hiPSC 介质。<ol>
<li>平衡补充剂和基础介质到室温 （RT）。确保补充已完全解冻。使用 0.22 μm 真空驱动过滤器混合 400 m...

<ol>
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使用人类 iPSC-CM 作为实验模型的关键步骤是：1） 生成高质量的心肌细胞 （CM），以确保一致的性能和可重现的结果;（ 1） 生成高质量的心肌细胞 （CM），2） 允许细胞在培养中成熟至少90天，以充分评估其表型;3） 进行电生理学研究，例如钙（Ca2+）瞬态测量，为人类iPSC-CM提供生理相关功能表征。我们开发了一种基于单层的分化方法，可生产高质量的心室状 iPSC-CM。我们的方法依赖于几个...

人类诱导多能干细胞衍生心肌细胞（iPSC-CMs）是一个有吸引力的基于人的平台，用于在体外模拟各种心脏疾病1、2、3、4、5、6、7、8。此外，iPSC-CM可用于预测患者对新药或现有药物的反应，筛选命中化合物，并开发新的个性化药物9，10。然而，尽管取得了显著进展，但在使用iPSC-CM11时，需要考虑一些限制和挑战。因此，改进心脏分化方案、提高iPSC-CMs效率和成熟以及产生特定心肌细胞亚型（心室、心房、心房和节点）的方法已被深入研究，并已导致许多文化策略克服这些障碍12、13、14、15。
尽管这些协议具有鲁棒性，但使用 iPSC-CM 的一个主要问题是，为了获得能够确保相同性能和可重复结果的高质量心肌细胞，需要重复和复杂的程序。重复性不仅在比较具有不同遗传背景的细胞系时，而且在重复同一细胞系的细胞和分子比较时都是至关重要的。细胞变异性（如 iPSC 密度的差异）可能会影响心脏分化，产生低产量和低质量的心肌细胞。这些细胞仍可用于执行不需要纯 VM 总体的实验（例如，执行 Ca2+瞬态测量时）。事实上，在进行电生理分析时，非CM不会跳动，无论是自发的，也不是电刺激的，所以很容易将它们排除在分析之外。然而，由于质量差，iPSC-CM 可以显示改变的电生理特性（例如，不规则的 Ca2+瞬态，低 Ca2+振幅），这些不是由于它们的遗传组成。因此，特别是在使用 iPSC-CM 对心脏病进行建模时，不要将劣质 CM 的结果与疾病表型混淆。在进行电生理学研究之前，需要仔细的筛选和排除过程。
该方法包括优化的协议，以生成高纯度和高质量的心肌细胞，并通过使用钙和收缩性采集和分析系统执行 Ca2+瞬态测量来评估其功能。该技术是一种简单而强大的方法，用于区分高效和低效率的 iPSC-CM 制剂，并提供更具有生理相关性的人类 iPSC-CM 表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-Actin, &#945;-Smooth Muscle antibody, Mouse monoclonal</td>
			<td>Sigma Aldrich</td>
			<td>A5228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti mouse</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti rabbit</td>
			<td>Invitrogen</td>
			<td>A21428</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27(-) insulin Supplement</td>
			<td>Gibco</td>
			<td>A18956-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI nuclear stain</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1X) + L- Glutamine + 15mM Hepes</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Ended Cell lifter, Flat blade and J-Hook</td>
			<td>Celltreat</td>
			<td>229306</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Multiwell Tissue Culture Plate, 6 well</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidic inline heater</td>
			<td>Live Cell Instrument</td>
			<td>IL-H-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2, AM</td>
			<td>Invitrogen</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC-qualified matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Matrigel Matrix</td>
		</tr>
		<tr>
			<td>hPSC media</td>
			<td>Gibco</td>
			<td>A33493-01</td>
			<td>StemFlex Basal Medium</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma Aldrich</td>
			<td>I0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Live cell imaging chamber</td>
			<td>Live Cell Instrument</td>
			<td>EC-B25</td>
			<td></td>
		</tr>
		<tr>
			<td>MLC-2A, Monoclonal Mouse Antibody</td>
			<td>Synaptic Systems</td>
			<td>311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Myocyte calcium and contractility system</td>
			<td>Ionoptix</td>
			<td>ISW-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Light Chain 2 Antibody, Rabbit Polyclonal (MLC2V)</td>
			<td>Proteintech</td>
			<td>10906-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid Flow Sterile Disposable Filter units with PES Membrane</td>
			<td>ThermoFisher</td>
			<td>124-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Calcium and Magensium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Glass Cover Slips</td>
			<td>Lab Scientific</td>
			<td>7807</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (-) D-glucose (1X)</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-lactate</td>
			<td>Sigma Aldrich</td>
			<td>L7022</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex Supplement</td>
			<td>Gibco</td>
			<td>A33492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazovivin</td>
			<td>Tocris</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution</td>
			<td>Boston Bioproducts</td>
			<td>BSS-355w</td>
			<td>Adjust pH at 7.2. Add 1.2mM Calcium Chloride</td>
		</tr>
	</tbody>
</table>

generation of ventricular-like hipsc-derived cardiomyocytes and high-quality cell preparations for calcium handling characterization

人类诱导多能干细胞衍生心肌细胞（iPSC-CMs）为研究钙（Ca2+）处理和信号通路以及高通量药物筛选和毒性检测的基本科学提供了宝贵的人类来源。在此，我们详细介绍了用于生成高质量 iPSC-CM 的方法，这些方法可以持续再现不同细胞系的分子和功能特征。此外，还介绍了一种方法，通过评估 Ca2+处理特性，可靠地评估其功能特性。低氧 （O2） 条件、 乳酸盐选择和长时间培养产生高纯度和高质量的心室状心肌细胞.与分离的成年大鼠心肌细胞 （ARCMs） 类似，3 个月大的 iPSC-CM 表现出更高的 Ca2+振幅、更快的 Ca2+再摄取率（衰变-tau），以及与第 30 天 iPSC-CM 相比，对 β-肾上腺素刺激的正热性反应。该策略在技术上简单、经济高效且可重现。它为心脏病建模和针对Ca2+处理蛋白的大规模药物筛查提供了一个强大的平台。

图1A中描述的协议生成了高度纯的心肌细胞，这些细胞在培养中具有随时间而获取心室/成人样表型。通过心房和心室肌苷调节光链2异种的免疫荧光染色评估，该协议产生的大多数细胞在诱导心脏分化后的第30天是MLC2A阳性，而MLC2V在同一时间点以更低的数量表示（图2A，顶部面板）。随着文化时间的增加（第 60 天和第 90 天），?...

Watch this Scientific Journal Video about Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization at JoVE.com

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

在这里，我们描述和验证一种持续产生强大的人类诱导多能干细胞衍生心肌细胞的方法，并描述其功能。这些技术可能有助于发展对信号通路的机械洞察力，为大规模药物筛查提供平台，并可靠地模拟心脏病。

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

在诱导干细胞衍生的心肌细胞时，可重复性是主要问题。使用这些方法，高纯度和高质量的心肌细胞可以从 iPS 中产生，用于功能研究。该技术的主要优点是，这是一种简单、可靠、经济高效、极其强大的方法，用于获得 iPSC 衍生心肌细胞的纯化、高质量制剂。对于细胞传传，用一毫升PBS冲洗细胞，不带钙和镁7至10分钟，然后用一毫升的传传介质替换PBS。使用细胞提升器，轻轻地从井底刮取细胞，并使用无菌的两毫升玻璃移液器机械地分离分离的细胞。当细胞被提升到显微镜下可见的小簇时，添加传通介质以一比六的比例分裂细胞。接下来，从六井板中吸出人类胚胎干细胞合格基质。然后将一毫升的传中和一毫升分离的细胞添加到每个井中，让细胞生长，直到它们达到70至80%的汇合，然后开始心脏分化。在分离 iPSC-CMs 之前至少 30 分钟，用 70% 乙醇擦拭单个玻璃盖滑，然后将盖滑入无菌六孔板的单个孔中。当盖板干燥后，将250至300微升的人类胚胎干细胞合格基质溶液直接添加到每个盖滑的中心，并将六个井板放在组织培养罩中。分离代谢选择的 iPSC-CM 添加一毫升无菌 0.25% trypsin 与 EDTA 到每个井在 35 摄氏度下孵育 5 分钟，并使用 1000 微升移液器机械分离细胞，直到实现单个细胞悬浮。将细胞在无菌的15毫升锥形管中拉，用每孔两毫升的RPMI 20介质清洗每孔。然后将洗涤液加入管子中，通过离心使细胞产生颗粒。以足够量的介质重新增加细胞，达到每250微升细胞浓度的3倍10至第五细胞。使用 1000 毫升移液器尖端分离细胞，直到溶液看起来是同质的。然后将250微升细胞吸进1000微升移液器尖端，并缓慢地将整体积溶液分配到一个人类胚胎干细胞合格基质编码盖滑上。当所有细胞都播种后，小心地将盘子放入细胞培养箱中过夜。第二天早上，轻轻将两毫升的D7介质加入到每井中。当选定的 iPSC-CM 达到至少三个月大时，用两个微升 Fura-2 AM 治疗细胞，在 37 摄氏度下 10 分钟，并打开钙分析系统电源。启动 ArcLamp，将泵中的管连接到相应的入口和出水口，将电子导线从刺激器连接到腔室。将腔室放在系统上。用 37 摄氏度加热 Tyrode 溶液填充流经流体内线加热器的 ProFusion 管，并调整相机和帧光圈尺寸，以最大限度地减少背景区域。将用 iPSC-CMs 种子的玻璃盖将滑入腔室，并轻轻地将 500 微升的 Tyrode 溶液直接添加到盖滑上。以每分钟 1.5 毫升的速率开始使用 Tyrode 的溶液，并使用电动刺激器以 1 赫兹场刺激的速度对 iPC-CMs 进行速度调整，每 4 毫秒 10 伏特，间隔 3 到 5 分钟。当所有染料都冲出腔室后，将观察窗口调整到盖滑的左上角区域，然后开始录制。收集 5 到 10 个峰值的一致流后，单击暂停以暂时停止录制，并在恢复录制之前将显微镜舞台移动到盖滑的对立面的相邻区域。在扫描整个盖片中的钙转性后，在 10 分钟内以相同的方式扫描数据，根据制造商的说明使用适当的荧光微量分析软件进行分析数据。该协议产生高度纯的心肌细胞，随着时间的推移，在培养中获得心室、成人样表型。根据心房和心室MLC2等构体免疫荧光染色评估，该协议产生的大多数细胞在心脏分化诱导后的第30天是心房MLC2异构体阳性。而心室MLC2等分形式在同一时间点以大量数字表示。随着培养时间的增加，观察到完全切换到 MLC2 等构形式。这些数据还通过流式细胞测量对心房和心室MLC2标记表达细胞的百分比进行定量确认。在第90天，iPSC-CMs中的钙振幅显著增加，与成年大鼠CMs的钙振幅相似。需要注意的是，污染非心脏细胞可能会影响人体 iPSC-CMs 在分化过程中的功能成熟。此外，在 iPSC-CMs 中记录钙瞬变时，有必要让细胞在恒定刺激下稳定在 37 摄氏度，持续至少 200 秒，并限制记录到特定时间窗口，以确保可重复的结果。请记住，iPSC 表现出均匀形态，并允许细胞在启动心脏分化之前增长到 70 至 80% 的汇合。未成熟的 iPS 衍生心肌细胞的纯质量可能显示改变的功能特征，这些特性不反映真正的表型，但可能被误解为患病的表型。因此，获得纯净和高质量的心肌细胞对于提供一致和可重复的结果非常重要。

Research

JoVE Journal

Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

处理棉鼠的研究为溶瘤病毒的临床前评估

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

指南生成和使用hiPSC派生的NPC的神经系统疾病的研究

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

分离和表征头颈部鳞状细胞癌亚群具有干细胞特性

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...