Name: sarcomere shortening of pluripotent stem cell-derived cardiomyocytes using fluorescent-tagged sarcomere proteins.
Uploaded: 2021-03-03T13:00:00
Description: Watch this Scientific Journal Video about Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins. at JoVE.com

我们感谢济池医科大学再生医学系的所有实验室成员提供有益的讨论和技术援助。这项研究得到了日本医学研究开发厅（AMED）的资助：JP18bm0704012 和 JP20bm0804018），日本科学促进会 （JSPS;JP19KK0219），以及日本流通协会（基础研究补助金）给H.U.

1. 小鼠多能干细胞的分化
<ol><li>维护鼠标 ES 单元格<ol>
<li>维护介质：混合 50 mL 的胎儿牛血清 （FBS）、5 mL 的 L-丙氨酸-L-谷氨酰胺、5 mL 的非必需氨基酸 （NEAA）、5 mL 的 100 mM 皮鲁瓦酸钠和 909 μl 的 55 mM 2-默卡普托乙醇与 450 mL 格拉斯哥最低基本介质 （GMEM）.。补充白血病抑制因子 （LIF）、CHIR-99021 和 PD0325901，最终浓度分别为 1000 U/mL、1 μM 和 3 μM。通过 0.22 μm 过滤器对介质进行消毒。</li>
		<li>FB...

<ol>
	<li>Mensah, G. A., Roth, G. A., Fuster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Global+Burden+of+Cardiovascular+Diseases+and+Risk+Factors.">The Global Burden of Cardiovascular Diseases and Risk Factors.</a> Journal of the American College of Cardiology. 74 (20), 2529-2532 (2019).</li><li>Wexler, R. K., Elton, T., Pleister, A., Feldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyopathy:+an+overview.">Cardiomyopathy: an overview.</a> American Family Physician. 79 (9), 778-784 (2009).</li><li>Parrotta, E. I., Lucchino, V., Scaramuzzino, L., Scalise, S., Cuda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Cardiac+Disease+Mechanisms+Using+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes:+Progress,+Promises+and+Challenges.">Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges.</a> International Journal of Molecular Sciences. , 30 (2020).</li><li>Carvajal-Vergara, X., 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=Patient-specific+induced+pluripotent+stem-cell-derived+models+of+LEOPARD+syndrome.">Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.</a> Nature. 465 (7299), 808-812 (2010).</li><li>Lan, F., 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=Abnormal+calcium+handling+properties+underlie+familial+hypertrophic+cardiomyopathy+pathology+in+patient-specific+induced+pluripotent+stem+cells.">Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.</a> Cell Stem Cell. 12 (1), 101-113 (2013).</li><li>Kim, C., 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=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494 (7435), 105-110 (2013).</li><li>Gramlich, M., 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=Antisense-mediated+exon+skipping:+a+therapeutic+strategy+for+titin-based+dilated+cardiomyopathy.">Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.</a> EMBO Molecular Medicine. 7 (5), 562-576 (2015).</li><li>Wang, G., 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=Modeling+the+mitochondrial+cardiomyopathy+of+Barth+syndrome+with+induced+pluripotent+stem+cell+and+heart-on-chip+technologies.">Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies.</a> Nature Medicine. 20 (6), 616-623 (2014).</li><li>Li, S., 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=Mitochondrial+Dysfunctions+Contribute+to+Hypertrophic+Cardiomyopathy+in+Patient+iPSC-Derived+Cardiomyocytes+with+MT-RNR2+Mutation.">Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation.</a> Stem Cell Reports. 10 (3), 808-821 (2018).</li><li>Cho, G. -. S., 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=Neonatal+Transplantation+Confers+Maturation+of+PSC-Derived+Cardiomyocytes+Conducive+to+Modeling+Cardiomyopathy.">Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy.</a> Cell Reports. 18 (2), 571-582 (2017).</li><li>Yang, X., 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=Tri-iodo-l-thyronine+promotes+the+maturation+of+human+cardiomyocytes-derived+from+induced+pluripotent+stem+cells.">Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.</a> Journal of Molecular and Cellular Cardiology. 72, 296-304 (2014).</li><li>Ribeiro, A. J. S., 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=Multi-Imaging+Method+to+Assay+the+Contractile+Mechanical+Output+of+Micropatterned+Human+iPSC-Derived+Cardiac+Myocytes.">Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes.</a> Circulation Research. 120 (10), 1572-1583 (2017).</li><li>Sewanan, L. R., Campbell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+sarcomeric+cardiomyopathies+with+human+cardiomyocytes+derived+from+induced+pluripotent+stem+cells.">Modelling sarcomeric cardiomyopathies with human cardiomyocytes derived from induced pluripotent stem cells.</a> The Journal of Physiology. 598 (14), 2909-2922 (2020).</li><li>Kopljar, I., 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=Chronic+drug-induced+effects+on+contractile+motion+properties+and+cardiac+biomarkers+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.</a> British Journal of Pharmacology. 174 (21), 3766-3779 (2017).</li><li>Riedl, 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nature Methods. 5 (7), 605-607 (2008).</li><li>Ribeiro, A. J. S., 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=Contractility+of+single+cardiomyocytes+differentiated+from+pluripotent+stem+cells+depends+on+physiological+shape+and+substrate+stiffness.">Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness.</a> Proceedings of the National Academy of Sciences. 112 (41), 12705-12710 (2015).</li><li>Roberts, B., 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=Fluorescent+Gene+Tagging+of+Transcriptionally+Silent+Genes+in+hiPSCs.">Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.</a> Stem Cell Reports. 12 (5), 1145-1158 (2019).</li><li>Chanthra, N., 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=A+Novel+Fluorescent+Reporter+System+Identifies+Laminin-511/521+as+Potent+Regulators+of+Cardiomyocyte+Maturation.">A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation.</a> Scientific Reports. 10 (1), 4249 (2020).</li><li>Ribeiro, M. C., 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=A+cardiomyocyte+show+of+force:+A+fluorescent+alpha-actinin+reporter+line+sheds+light+on+human+cardiomyocyte+contractility+versus+substrate+stiffness.">A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.</a> Journal of Molecular and Cellular Cardiology. 141, 54-64 (2020).</li><li>Yamanaka, S., Zahanich, I., Wersto, R. P., Boheler, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Proliferation+of+Monolayer+Cultures+of+Embryonic+Stem+(ES)+Cell-Derived+Cardiomyocytes+Following+Acute+Loss+of+Retinoblastoma.">Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma.</a> PLoS ONE. 3 (12), 3896 (2008).</li><li>Miyazaki, T., Isobe, T., Nakatsuji, N., Suemori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Adhesion+Culture+of+Human+Pluripotent+Stem+Cells+Using+Laminin+Fragments+in+an+Uncoated+Manner.">Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.</a> Scientific Reports. 7 (1), 41165 (2017).</li><li>Prasad, K. -. M. R., Xu, Y., Yang, Z., Acton, S. T., French, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cardiomyocyte-specific+gene+expression+following+systemic+injection+of+AAV:+in+vivo+gene+delivery+follows+a+Poisson+distribution.">Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution.</a> Gene Therapy. 18 (1), 43-52 (2011).</li><li>Arakawa, H., 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=Protein+evolution+by+hypermutation+and+selection+in+the+B+cell+line+DT40.">Protein evolution by hypermutation and selection in the B cell line DT40.</a> Nucleic Acids Research. 36 (1), e1 (2007).</li><li>Konno, A., Hirai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+whole+brain+transduction+by+systemic+infusion+of+minimally+purified+AAV-PHP.eB.">Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB.</a> Journal of Neuroscience Methods. 346, 108914 (2020).</li><li>Anzai, T., et al., Yoshida, Y., 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=Generation+of+Efficient+Knock-in+Mouse+and+Human+Pluripotent+Stem+Cells+Using+CRISPR-Cas9.">Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9.</a> Methods of Molecular Biology. , (2020).</li><li>Ahmed, R. E., Anzai, T., Chanthra, N., Uosaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Brief+Review+of+Current+Maturation+Methods+for+Human+Induced+Pluripotent+Stem+Cells-Derived+Cardiomyocytes.">A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes.</a> Frontiers in Cell and Developmental Biology. 8, 178 (2020).</li><li>Moretti, A., 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=Patient-specific+induced+pluripotent+stem-cell+models+for+long-QT+syndrome.">Patient-specific induced pluripotent stem-cell models for long-QT syndrome.</a> The New England Journal of Medicine. 363 (15), 1397-1409 (2010).</li><li>Flores, L. R., Keeling, M. C., Zhang, X., Sliogeryte, K., Gavara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact-GFP+alters+F-actin+organization,+cellular+morphology+and+biophysical+behaviour.">Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour.</a> Scientific Reports. 9 (1), 3241 (2019).</li><li>Sparrow, A. 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=Measurement+of+Myofilament-Localized+Calcium+Dynamics+in+Adult+Cardiomyocytes+and+the+Effect+of+Hypertrophic+Cardiomyopathy+Mutations.">Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.</a> Circulation Research. 124 (8), 1228-1239 (2019).</li></ol>

PSC-CMs具有巨大的潜力，可以作为体外 平台来模拟心脏病和测试药物的效果。然而，必须首先建立一种准确、统一的方法来评估 PSC-CM 功能。大多数功能测试都与 PSC-CMs 配合使用，例如电生理学、钙瞬态和代谢26，而第一批患者衍生的 PSC-CM 研究之一是关于长 QT 综合征27。然而，收缩性是心肌细胞最重要的功能之一，仍然难以评估。成人心肌细胞，肉瘤缩?...

心血管疾病是全世界1人死亡的主要原因，心肌病是心脏相关死亡的第三大原因。心肌病是影响心脏肌肉的一组疾病。诱导多能干细胞（iPS）细胞的最新发展以及iPS细胞对心肌细胞（PSC-CMs）的定向分化，为研究作为心肌病体外模型的患者基因组心肌细胞打开了大门。这些细胞可用于了解心脏病的病理生理学，阐明其分子机制，并测试不同的治疗候选者3。因此，产生了大量的兴趣，因此，产生了患者衍生的iPS细胞（例如，肥大性心肌病[HCM]4，5，心律失常右心室心肌病[ARVC]6，扩张性心肌病[DCM]7，和线粒体相关心肌病8，9）。因为心肌病的特征之一是肉瘤功能障碍和紊乱，因此需要一种能统一测量肉瘤功能的有效工具。
肉瘤缩短是评估肉瘤功能和从动物模型和人类衍生的成人心肌细胞收缩性最广泛使用的技术。要执行肉瘤缩短，需要在相对比下可见的发达肉瘤。然而，PSC-CMs培养的体外显示器不发达和混乱的肉瘤，因此，不能用于正确测量肉瘤缩短10。这种难以正确评估PCS-CMs收缩性的问题阻碍了它们作为体外评估心脏功能的平台的使用。为了间接评估PCS-CMs收缩性，原子力显微镜、微柱阵列、牵引力显微镜和阻抗测量被用来测量这些细胞对周围环境的运动效果。更复杂和侵入性较小的视频显微镜记录的实际细胞运动（例如，SI8000从索尼）可以用来替代评估其收缩性，但是，这种方法不能直接测量肉瘤运动或力生成动能14。
为了直接测量PCC-CMs中的肉瘤运动，新的方法，如荧光标记到肉瘤蛋白，正在出现。例如，Lifeact 用于标记丝状活性素 （F-actin） 来测量肉瘤运动15、16。转基因iPS细胞是另一种选择标记肉瘤蛋白（例如，α-actinin[ACTN2]和Myomesin-2[MYOM2]）由荧光蛋白17，18，19。
在本文中，我们描述了如何使用 Myom2-TagRFP（小鼠胚胎干细胞 [ES] 细胞） 和 ACTN2-mCherry（人类 iPS 细胞）执行测量肉瘤缩短的延时成像。我们还表明，有图案的文化有助于肉瘤的排列。此外，我们使用腺相关病毒 （AAV） 描述肉瘤标记的替代方法，该病毒可广泛应用于患者衍生的 iPS 细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145-25</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (55mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methacryloyloxyethyl phosphorylcholine (MPC) polymer,</td>
			<td>NOF Corp.</td>
			<td>LIPIDURE-CM5206</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fujifilm wako</td>
			<td>166-04836</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm imaging dish with a polymer coverslip (&#181;-Dish 35 mm, high)</td>
			<td>ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>AAVproR Helper Free System (AAV6) 
			(vectors; pHelper, pRC6, pAAV-CMV-Vector)</td>
			<td>Takara</td>
			<td>6651</td>
			<td></td>
		</tr>
		<tr>
			<td>ACTN2-mCherry (AR12, AR21) hiPSCs</td>
			<td>N.A.</td>
			<td></td>
			<td>We inserted IRES-puromycin resistant casette to 3&#39; UTR of TNNT2 locus and mCherry around the stop codon of ACTN2 in 610B1 hiPSC line, following a method describe elsewhere (Anzai, Methods Mol Biol, in press)</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, minus insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A18956-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (50X), minus Vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase (25 U/&#181;L)</td>
			<td>Merck Millipore</td>
			<td>70746</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin S Hydrochloride</td>
			<td>Fujifilm wako</td>
			<td>029-18701</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4, Human, Recombinant,</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>C59, Wnt Antagonist (WntC59)</td>
			<td>abcam</td>
			<td>ab142216</td>
			<td></td>
		</tr>
		<tr>
			<td>CAD drawing software,</td>
			<td>Robert McNeel and Associates, WA, USA</td>
			<td>Rhinoceros 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal ultrafiltration unit (100k MWCO), Vivaspin-20</td>
			<td>Sartorius</td>
			<td>VS2042</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman</td>
			<td>13122</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium etchant</td>
			<td>Nihon Kagaku Sangyo Co., Ltd., Japan</td>
			<td>N14B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium mask coated with AZP1350</td>
			<td>Clean Surface Technology Co., Japan</td>
			<td>CBL2506Bu-AZP</td>
			<td></td>
		</tr>
		<tr>
			<td>Dr. GenTLE Precipitation Carrier (20mg/mL Glycogen, 3 M Sodium Acetate (pH 5.2))</td>
			<td>Takara</td>
			<td>9094</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D6429-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) - high glucose, without sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (99.5)</td>
			<td>Fujifilm wako</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Moregate</td>
			<td>59301104</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10, Human, Recombinant,</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>345-FG-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor(basic), human, recombinant</td>
			<td>Fujifilm wako</td>
			<td>060-04543</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin powder</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow Minimum Essential Medium (GMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>G5154-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GLASS BOTTOM culture plates</td>
			<td>MatTek</td>
			<td>P24G-1.5-13-F/H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11765-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440-061</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanine-L-glutamine (GlutaMAX Supplement, 200mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Ascorbic Acid Sodium Salt</td>
			<td>Fujifilm wako</td>
			<td>196-01252</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin-511 E8 fragment (LN511-E8, iMatrix-511)</td>
			<td>Nippi</td>
			<td>892012</td>
			<td></td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>Union Optical Co., Ltd., Japan</td>
			<td>PEM-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Maskless lithography tool</td>
			<td>NanoSystem Solutions, Inc., Japan</td>
			<td>D-Light DL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45 &#181;m, PVDF (0.45-&#181;m filter)</td>
			<td>Merck Millipore</td>
			<td>SLHVR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Myom2-RFP (SMM18)</td>
			<td>N.A.</td>
			<td></td>
			<td>Developed in our previous paper (Chanthra, Sci Rep, 2020)</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 V3 digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C13440-20CU</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Stemgent</td>
			<td>04-0006-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sansei medical co. Ltd</td>
			<td>01-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl alcohol (25:24:1)</td>
			<td>Nippon Gene</td>
			<td>311-90151</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) elastomer</td>
			<td>Dow Corning Corp., MI, USA</td>
			<td>SILPOT 184</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylenimine MAX (MW. 40,000)</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive photoresist developer</td>
			<td>Tokyo Ohka Kogyo Co., Ltd., Japan</td>
			<td>NMD-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25742</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Takara</td>
			<td>9034</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>Fujifilm wako</td>
			<td>166-23153</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Mouse/Rat Activin A Protein</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant trypsin-like protease (rTrypsin; TrypLE express)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604-039</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Matsuzaki Seisakusyo Co., Ltd., Japan</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Mikasa Co., Ltd., Japan</td>
			<td>MS-A100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spininng confocal microscopy</td>
			<td>Oxford Instruments</td>
			<td>Andor Dragonfly Spinning Disk System</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSure LIF, Mouse, recombinant, Solution (10^6U)</td>
			<td>Fujifilm wako</td>
			<td>195-16053</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3010</td>
			<td>Kayaku Advanced Materials, Inc., MA, USA</td>
			<td>SU-8 3010</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Kayaku Advanced Materials, Inc., MA, USA</td>
			<td>SU-8 developer</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA</td>
			<td>Nippon Gene</td>
			<td>314-90021</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor-A165(VEGF), Human, recombinant</td>
			<td>Fujifilm wako</td>
			<td>226-01781</td>
			<td></td>
		</tr>
	</tbody>
</table>

sarcomere shortening of pluripotent stem cell-derived cardiomyocytes using fluorescent-tagged sarcomere proteins.

多能干细胞衍生心肌细胞（PSC-CMs）可以同时从胚胎干细胞和诱导多能干细胞（ES/iPS）细胞中产生。这些细胞为心脏病建模提供了有希望的来源。对于心肌病，肉瘤缩短是标准生理评估之一，用于成人心肌细胞检查其疾病表型。然而，现有的方法不适合评估PCS-CM的收缩性，因为这些细胞的肉瘤发育不全，在相对比显微镜下是看不见的。为了解决这个问题，并用PCC-CMs进行肉瘤缩短，使用了荧光标记的肉瘤蛋白和荧光活成像。细 Z 线和 M 线分别位于肉瘤的两端和中心。Z线蛋白 - α-Actinin （ACTN2）、电传素 （TCAP） 和与 ACTIN 相关的 LIM 蛋白 （PDLIM3） 和一种 M 线蛋白-肌素-2 （Myom2） 被标记为荧光蛋白。这些标记的蛋白质可以从内源性等位基因作为敲击或腺相关病毒 （AAV） 表达。在这里，我们介绍了将小鼠和人类多能干细胞分化到心肌细胞、产生 AAV 以及执行和分析活成像的方法。我们还描述了为 PSC-CM 的图案培养物生产聚二甲基硅氧烷 （PDMS） 邮票的方法，这有助于分析带有荧光标记的蛋白质的肉瘤缩短。为了评估肉瘤缩短，在电刺激（0.5-1 Hz）下，以高帧速率（每秒50-100帧）记录了跳动细胞的延时图像。为了分析细胞收缩过程中的肉瘤长度，录制的延时图像受制于 SarcOptiM，这是 ImageJ/Fiji 的插件。我们的战略为研究 PSC-CMs 中的心脏病表型提供了一个简单的平台。

使用敲击 PSC-CMs 记者线测量肉瘤缩短。沙科默标签的 PSC-CM 用于测量肉瘤缩短。线条表达 Myom2 - rfp 和 Actn2 - mcherry 从内源性洛西。TagRFP 入Myom2 ，编码 M 蛋白本地化到 M 线，而 mCherry 被敲入ACTN2 ，编码α - 阿克蒂宁，本地化到 Z 线 18 ， 25 。获得延时图像，并用于确定肉瘤缩短，如图1和...

Watch this Scientific Journal Video about Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins. at JoVE.com

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

该方法可用于使用多能干细胞衍生心肌细胞与荧光标记肉瘤蛋白来检查肉瘤缩短。

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

由于多能干细胞衍生的结核细胞发育不全，肉瘤功能难以评估。使用荧光标记肉瘤蛋白，我们现在可以获得肉瘤缩短。我们可以使用干细胞衍生的心肌细胞和荧光标记肉瘤蛋白来可视化肉瘤，并在体外评估其功能。使用基于 AAV 的转导，我们可以将该方法扩展到任何患者衍生的诱导多能干细胞。这种方法可以扩展到心肌病治疗的发展，因为它可以用来直接评估与疾病相关的肉瘤功能障碍在体外。这种方法在心脏病学领域很有用，包括儿科心脏病学研究。然而，它也可以应用于骨骼肌功能障碍的研究，如肌病。在减去四分化诱导的第二天，涂上一个六井板，每平方厘米的层压素-511 E8在PBS中稀释0.5微克，在37摄氏度和5%的二氧化碳下孵育30分钟。在潜伏接近尾声时，在37摄氏度下用重组试金素治疗人类诱导的多能干细胞三分钟。当细胞分离后，将细胞收获到中等，辅以10%FBS进行计数，并在离心后以每两毫升AK02N介质的1.25倍10至5个细胞重新使用细胞，辅以层压素-511 E8和ROCK抑制剂。接下来，从六井板的每口井中吸出涂层溶液，并将两毫升细胞播种到每口井中。将板返回到细胞培养孵化器。四天后，细胞达到80%的汇流。用两毫升RPMI加B27代替每口油井中的超高纳坦，每口井不加胰岛素介质，并辅以葛兰素-3抑制剂，以诱导分化。经过两天的分化，轻轻地用两毫升RPMI加B27代替介质，而没有胰岛素介质，每口井补充豪猪抑制剂。细胞应该达到100%的汇流。在分化的第四天，用两毫升RPMI加B27代替超高纳特，每口井不穿胰岛素。细胞应该保持高密度，有些可能已经开始漂浮。在分化的第7天和第9天，用两毫升RPMI加B27代替每口井中的超高纳坦，用胰岛素补充纯霉素，用于选择性多能干细胞衍生心肌细胞培养。此时，应在培养物中观察跳动细胞。要建立一种图案多能干细胞衍生心肌细胞培养，首先使用修补胶带去除定制PDMS邮票表面的任何灰尘，并短暂地将邮票浸入70%乙醇中。使用空气除尘器从邮票表面去除乙醇，并用5至10微升0.5%MPC聚合物和乙醇处理表面。当聚合物完全干燥时，将邮票放在35毫米成像盘的聚合物盖片上，并在邮票上放置重量。10 分钟后，使用光显微镜确认图案已转移到盖唇上，并用 PBS 洗两次盘和盖唇。第二次洗涤后，将盖唇涂上每平方厘米0.5至1微克的层压素-511 E8稀释在0.1%明胶中，在室温下孵育2至4小时。在分化的第10天，用PBS清洗细胞两次，然后用每口井一毫升重组试丁香在37摄氏度下治疗三到五分钟。计算后，以每毫升RPMI加B27的2.5至5倍10至5倍的细胞，辅以胰岛素，辅以纯霉素浓度，将1毫升细胞重新播种到板上，在37摄氏度下过夜孵育。第二天早上，用新鲜的RPMI加B27代替超高纳坦，用胰岛素补充纯霉素，将细胞返回细胞培养孵化器，每周刷新介质两到三次，直到21至28天进行成像。对于 PCCSM 培养物中肉瘤缩短的延时成像，请将油涂抹到荧光聚焦显微镜的 100 倍客观镜头中，并在相关软件中选择实时成像条件。要获得良好的代表性数据，请选择最高帧速率，设置快门以打开，并在采集区域的作物中应用四乘四的装箱，以便在延时成像期间实现图像之间的最短间隔。如果细胞的跳动率较低，则通过电场刺激唤起细胞，并开始延时记录，注意成像过程中成像场保持对焦。在成像期结束时，将延时图像保存到适当的文件夹中。要分析延时图像，请将一系列延时图像打开到 ImageJ 中，并调整图像的亮度和对比度，直到可以清楚地观察到肉瘤图案。在"更多工具"菜单中，选择 SarcOptiM，并按下控制移位 P 和工具栏上的一微米按钮，以使用说明校准程序。然后划出一条线穿过肉瘤区域，用于测量肉瘤缩短，然后单击单细胞 AVI 开始肉瘤缩短分析。肉瘤标签、图案多能干细胞衍生心肌细胞的延时成像可用于测量不同时间点的肉瘤缩短。为了克服在模式多能干细胞衍生心肌细胞培养中通常观察到的肉瘤杂乱无章，特定的PDMS邮票可用于培养条纹图案的多能干细胞衍生心肌细胞，与在非模式区域培养的细胞相比，它促进拉长细胞形状和更有条理的肉瘤模式。与非模式培养细胞相比，模式培养还促进更好的细胞收缩，并提供平滑的肉瘤长度轮廓。AAV 转导模式多能干细胞衍生心肌细胞在转导后三天内就沿着模式多能干细胞衍生心肌细胞表达肉瘤 GFP 信号，也可用于测量肉瘤收缩轮廓。此外，在转导过程中添加抗爆霉素基因也可用于促进模式多能干细胞衍生心肌细胞选择，使其纯度超过 90%，以便于更轻松、更高质量的图像分析，因此必须识别具有线性移动的肉瘤区域，并以最高帧速率获取图像。利用这种方法，我们可以评估肉瘤蛋白成熟的作用，以研究来自疾病特异性患者衍生的iPS细胞的实验室心肌细胞中的肉瘤功能障碍。

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 ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

使用hiPSC衍生心肌细胞的缺血性心脏病模型和基于视频的心肌细胞收缩比较

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

从人多能干细胞中定向诱导视网膜类器官

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...