Name: analyzing the &alpha;-actinin network in human ipsc-derived cardiomyocytes using single molecule localization microscopy
Uploaded: 2020-11-03T13:00:00
Description: Watch this Scientific Journal Video about Analyzing the ÃŽÂ±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy at JoVE.com

这项研究得到了欧盟结构基金（ESF/14-BM-A55-0024/18）的支持。此外，H.L.还得到罗斯托克大学医学中心（889001和889003）和约瑟夫和克林茨基金会（T319/29737/2017）的 FORUN 方案的支持。C.I.L. 得到罗斯托克大学医学中心临床科学家计划的支持。R.D 由 DFG （DA1296/6-1）、DAMP 基金会、德国心脏基金会 （F/01/12） 和 BMBF （VIP+ 00240） 支持。
我们感谢玛德琳·巴奇在 iPSC 细胞培养和心脏分化方面给予的技术支持。

本议定书中涉及新生儿和成年小鼠的所有步骤都按照罗斯托克大学医学中心动物护理的道德准则执行。
1. iPSC衍生心肌细胞的培养和分离
<ol><li>使用 2D 单层方法区分 hiPSC-VM 25 天，如前面所述。</li>
	<li>预热分离介质至37°C，支持中室温度。</li>
	<li>用 PBS 清洗细胞两次。</li>
	<li>将预预热分离介质添加到细胞中，在37°C和5%CO2下孵育12分钟。</li>
	<li>向?...

<ol>
	<li>McAloon, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+face+of+cardiovascular+disease+2000-2012:+An+analysis+of+the+world+health+organisation+global+health+estimates+data.">The changing face of cardiovascular disease 2000-2012: An analysis of the world health organisation global health estimates data.</a> International Journal of Cardiology. 224, 256-264 (2016).</li><li>Kussauer, S., David, R., Lemcke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hiPSCs+Derived+Cardiac+Cells+for+Drug+and+Toxicity+Screening+and+Disease+Modeling:+What+Micro-+Electrode-Array+Analyses+Can+Tell+Us.">hiPSCs Derived Cardiac Cells for Drug and Toxicity Screening and Disease Modeling: What Micro- Electrode-Array Analyses Can Tell Us.</a> Cells. 8 (11), 1331 (2019).</li><li>Yl&#228;-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC-Derived+Cardiomyocytes+Taken+to+Rescue+Infarcted+Heart+Muscle+in+Coronary+Heart+Disease+Patients.">iPSC-Derived Cardiomyocytes Taken to Rescue Infarcted Heart Muscle in Coronary Heart Disease Patients.</a> Molecular Therapy. 26 (9), 2077 (2018).</li><li>Kadota, S., Shiba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+Stem+Cell-Derived+Cardiomyocyte+Transplantation+for+Heart+Disease+Treatment.">Pluripotent Stem Cell-Derived Cardiomyocyte Transplantation for Heart Disease Treatment.</a> Current Cardiology Reports. 21 (8), 73 (2019).</li><li>Jiang, Y., Park, P., Hong, S. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+adolescence:+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Circulation Research. 114 (3), 511-523 (2014).</li><li>Bedada, F. B., Wheelwright, M., Metzger, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cronos+Titin+Is+Expressed+in+Human+Cardiomyocytes+and+Necessary+for+Normal+Sarcomere+Function.">Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.</a> Circulation. 140 (20), 1647-1660 (2019).</li><li>Fornasiero, E. F., Opazo, F., Fornasiero, E. 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体外功能 iPSC 衍生 CM 的生成对于再生疗法、疾病建模和药物筛选平台的发展非常重要。然而，这些CM的成熟度不足是心血管研究20的主要障碍。在这方面，需要高分辨率成像技术，以便监测 iPSC 衍生 CM 的结构成熟状态。同时，超分辨率显微镜可以是一个有价值的工具，可以精确分析特定蛋白质的功能，需要适当的沙康，最近证明的滴定和肌蛋白10，21，22。,<...

心血管疾病（CVD）（如心肌梗塞或心肌病）仍然是西方世界的主要死因。由于人类心脏只有不良的再生能力，因此需要制定战略来促进心血管疾病的恢复。这包括细胞替代疗法，以补充丢失的心肌细胞（CM），以及开发新的抗心律失常药物，以高效和安全的药物干预。诱导多能干细胞（iPSC）已被证明是一个有希望的细胞来源，无限代人类CM在体外，适合再生疗法，疾病建模，并用于药物筛选测定,2，3，43的发展。2
虽然存在许多不同的心脏分化协议，iPSC衍生的CM仍然缺乏某些表型和功能方面，阻碍体外和体内应用5,5，6。除了电生理、代谢和分子变化，心脏成熟过程涉及沙状物的结构组织，这是力生成和细胞收缩所需的基本单位。虽然成人CMs表现出组织良好的收缩装置，iPSC衍生的CMs通常表现出不一和的沙康丝，与收缩能力降低和改变收缩动力学8,8，9。与显示单轴收缩模式的成熟CM相比，未成熟CM中的迷失方向结构导致整个细胞的径向收缩或促进收缩焦点9、10,的出现。
为了改善心脏成熟，已经应用了多种方法，包括3D细胞培养方法，电和机械刺激，以及使用细胞外基质模仿体内条件11，12，13。11,12,13为了评估这些不同培养条件的成功和效率，需要技术来监测和估计 iPSC CM 的结构成熟程度，例如，通过微观技术。与传统共体成像相比，光激活定位显微镜 （PALM） 的分辨率高出约 10 倍。这种技术反过来允许更准确的分析，检测甚至微妙的变化的细胞结构14。考虑到PALM成像的高分辨率，该方法的总体目标是通过精确测定z-Disc厚度和沙康长度，对psC衍生的CMs中沙科勒成熟度进行微观评估。在以前的研究中，这些结构特征已被证明是评估心脏成熟度15的适当参数。例如，与野生细胞16相比，缺乏全长肌营养不良素的病症 iPSC-CM 表现出减少的沙康长度和 z 波段宽度。同样，测量单个沙形病的长度，以调查地形图线索对心脏发育的影响16。因此，我们运用这种方法对 iPSC-CM 中沙康器网络的结构成熟度进行了定量测量，从而测量了沙康卡长度和 z-disc 厚度。

<table>
	<tbody>
		<tr>
			<td>human iPSC cell line</td>
			<td>Takara</td>
			<td>Y00325</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well Glass Bottom</td>
			<td>ibidi</td>
			<td>80827</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5ml eppendorf tube</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiomyocyte Dissociation Kit</td>
			<td>Stem Cell Technologies</td>
			<td>05025</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma Aldrich</td>
			<td>C40-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctatetraene</td>
			<td>Sigma Aldrich</td>
			<td>138924-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteamine</td>
			<td>Sigma Aldrich</td>
			<td>30070-10g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Ca2+ and Mg2+</td>
			<td>Thermo Fisher</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2-Goat anti-Mouse IgG Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21237</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image processing software (Image J)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Carl Roth</td>
			<td>X997.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 780 ELYRA PS.1 system</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>8187150100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyranose oxidase</td>
			<td>Sigma Aldrich</td>
			<td>P4234-250UN</td>
			<td></td>
		</tr>
		<tr>
			<td>sarcomeric &#945;-actinin antibody [EA-53]</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile water</td>
			<td>Carl Roth</td>
			<td>3255.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the &alpha;-actinin network in human ipsc-derived cardiomyocytes using single molecule localization microscopy

iPSC衍生心肌细胞的成熟是它们在再生治疗、药物测试和疾病建模中应用的一个关键问题。尽管开发了多种分化方案，但类似成人表型的 iPSC 心肌细胞的生成仍然具有挑战性。心肌细胞成熟的一个主要方面是形成组织良好的沙科动物网络，以确保高收缩能力。在这里，我们提出了一种基于超分辨率的方法，用于对心肌细胞中α-actinin网络进行半定量分析。利用光激活定位显微镜，对从新生儿组织分离的 iPSC 衍生心肌细胞和心脏细胞的沙康长度和 z-disc 厚度进行了比较。同时，我们证明了适当的成像条件对于获得可靠数据的重要性。结果表明，该方法适合定量监测具有高空间分辨率的心脏细胞的结构成熟度，能够检测到沙康组织甚至细微的变化。

为了估计CM、新生儿、完全成熟的成人和 iPSC CM 的结构成熟程度，最初用 α-actinin 抗体标记 CM 以可视化沙康网络。在PALM采集后，对图像进行了重建，并使用基于插件的图像处理软件测量了z-disc厚度，以自动检测单个细丝的宽度。通过测量两个相邻强度峰之间的距离（对应于相邻的长丝）计算了 Sarcomere 长度。 图 1 显示了 iPSC 派生 CM 中对 sarcomere 组织的评估。
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Watch this Scientific Journal Video about Analyzing the ÃŽÂ±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy at JoVE.com

Analyzing the ÃƒÅ½Ã‚Â±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

形成适当的沙康网络对于 iPSC 衍生心肌细胞的成熟非常重要。我们提出了一种基于超分辨率的方法，允许对干细胞衍生心肌细胞的结构成熟进行定量评估，以改善促进心脏发育的培养条件。

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

该方法可用于对 iPSC 衍生心肌细胞中沙体成熟进行定量评估。使用这种基于分辨率的超级方法，可以检测出沙康组织中的细微变化，这在传统的共声成像中是不可能的。使用前至少三小时，打开显微镜，将样品置于室温。当显微镜准备就绪时，将 300 微升 PALM 成像缓冲液添加到标记的细胞井中，并将腔室幻灯片插入显微镜的舞台支架中。选择 1.57 NA 100X 油目标，并在成像软件中使用 PALM 模式并激活 TIRF 设置。将帧数设置为 5，000 至 10，000，将 UV 激光功率设置为 0.1%，将 647 激光器设置为 0.2%，将增益级别设置为 50 至 100。设置所有采集参数后，打开激光照明并选择目标单元。要获取图像，请将 647 激光功率提高至 100%，将增益减小到零。漂白目标细胞约五秒钟，然后将增益增加至50，然后启动PALM图像采集。对于PALM数据的重建，在采集结束时，打开 ImageJ 中的数据并打开雷雨插件。在插件中选择运行分析并打开摄像机设置菜单。输入像素大小和电磁增益。在运行分析菜单中，将B样条顺序设置为三，B样条比例为二，峰值强度阈值为stfwave。f1，拟合半径为三，初始西格玛为1.6，放大到5，更新频率为50，横向移到2，然后单击"确定"。重建后，在绘图直方图菜单中选择西格玛，并使用矩形工具选择感兴趣的区域，但不包括可能的工件。将感兴趣区域添加到筛选器中，并将小于 25 的不确定点添加到感兴趣值区域。在"删除重复项"选项卡中，输入 10 纳米的距离阈值。在合并选项卡中，将最大距离设置为 20，将每个分子的最大帧设置为零，将最大关闭帧设置为 1。在漂移校正选项卡中，选择交叉关联，将条柱数和放大率设置为 5，然后保存最终的 PALM 图像并导出后处理数据。要分析沙康长度，请将重建的感兴趣的PALM图像导入 ImageJ，并使用线条工具在垂直于 Z-disc 的选定 sarcomere 结构之间绘制一条线，以测量行为丝之间的最短距离。在分析菜单中，选择绘图轮廓并获取两个峰值之间的长度。要分析 Z-disc 厚度，请将重建的 PALM 图像转换为八位模式图像，然后打开 Ridge 检测插件。将线宽设置为 20，将高对比度设置为 230，将低对比度设置为 10，将西格玛设置为 0.79，将下半阈值设置为 25.84，将最小线长设置为 20。选择估计宽度、延伸线和显示结果，然后单击"确定"并使用结果表中的均线宽度进行进一步分析。iPSC衍生的心肌细胞和新生儿细胞表现出类似的α行为素模式与不规则的不安排的沙康体结构。定量评估表明，两组细胞之间几乎相同的α作用素丝的长度和厚度，表明 iPSC 衍生的心肌细胞发育过早。相比之下，成年成熟心肌细胞表现出常规的沙康网络，具有略大的沙康长度和减少的Z-disc厚度。比较传统的共体成像和PALM，发现沙康长度没有显著差异。然而，当 iPSC 心肌细胞进行 PALM 成像时，检测到 Z 盘厚度的显著降低。应用 PALM 时观察到分辨率的增益，相应的强度图支持。值得注意的是，与在最佳成像条件下捕获的结构相比，使用低质量缓冲器获得的沙康结构似乎更厚。数据准确性的缺乏是由于荧光团的闪烁性能降低，导致每个定位事件检测到的光子更少。此外，定位精度降低，降低了重建的PALM图像的整体分辨率。此外，样品漂移会影响荧光分子的精确定位，从而导致图像模糊。为了获得正确的PALM图像，让整个成像系统的热平衡以避免成像过程中样品过度漂移是非常重要的。按照这个程序，其他细胞结构，如线粒体可以由PALM成像，以获得心肌细胞成熟的其他参数。

Research

JoVE Journal

Biology

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Single-Molecule Imaging of Nuclear Transport

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

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

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Studying DNA Looping by Single-Molecule FRET

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...