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

We would like to acknowledge all the lab members in the Division of Regenerative Medicine at the Jichi Medical University for the helpful discussion and technical assistance. This study was supported by the grants from the Japan Agency for Medical Research and Development (AMED; JP18bm0704012&#160;and JP20bm0804018), the Japan Society for the Promotion of Science (JSPS; JP19KK0219), and the Japanese Circulation Society (the Grant for Basic Research) to H.U.

1. Differentiation of mouse pluripotent stem cells
<ol>
	<li>Maintenance of mouse ES cells
	<ol>
		<li>Maintenance medium: Mix 50 mL of fetal bovine serum (FBS), 5 mL of L-alanine-L-glutamine, 5 mL of non-essential amino acid (NEAA), 5 mL of 100 mM Sodium Pyruvate, and 909 &#956;l of 55 mM 2-Mercaptoethanol with 450 mL of Glasgow Minimum Essential Medium (GMEM). Supplement Leukemia inhibitory factor (LIF), CHIR-99021, and PD0325901 at a final concentration of 1000 U/mL, 1 &#956;M, and 3 &#956;M, respectively. Sterilize...

<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 have great potential to be utilized as an&#160;in vitro platform to model heart disease and to test the effects of drugs. Nevertheless, an accurate, unified method to assess PSC-CMs functions must first be established. Most of functional tests work with PSC-CMs, e.g., electrophysiology, calcium transient, and metabolism26, and one of the first patient-derived PSC-CM studies was about long-QT syndrome27. However, contractility, one of the most important func...

Cardiovascular diseases are the leading cause of mortality worldwide1 and cardiomyopathy represents the third cause of cardiac-related deaths2.Cardiomyopathy is a collective group of diseases that affect cardiac muscles. The recent developments of induced pluripotent stem (iPS) cells and the directed-differentiation of iPS cells toward cardiomyocytes (PSC-CMs) have opened the door for studying cardiomyocytes with patient genome as an in vitro model of cardiomyopathy. These cells can be used to understand the pathophysiology of cardiac diseases, to elucidate their molecular mechanisms, and to test different therapeutic candidates3. There is a tremendous amount of interest, thus, patient-derived iPS cells have been generated (e.g., hypertrophic cardiomyopathy [HCM]4,5, arrhythmogenic right ventricular cardiomyopathy [ARVC]6, dilated cardiomyopathy [DCM]7, and mitochondrial-related cardiomyopathies8,9). Because one of the characteristics of cardiomyopathy is the dysfunction and disruption of sarcomeres, a valid tool that uniformly measures sarcomere function is needed.
Sarcomere shortening is the most widely used technique to assess sarcomere function and the contractility of adult cardiomyocytes derived from animal models and humans. To perform sarcomere shortening, well-developed sarcomeres that are visible under phase-contrast are required. However, PSC-CMs cultured in vitro display underdeveloped and disorganized sarcomeres and, therefore, are unable to be used to properly measure sarcomere shortening10. This difficulty to properly assess the contractility of PSC-CMs hinders their usage as a platform to assess cardiac functions in vitro. To assess PSC-CMs contractility indirectly, atomic force microscopy, micro-post arrays, traction force microscopy, and impedance measurements have been used to measure the effects of the motion exerted by these cells on their surroundings11,12,13. More sophisticated and less invasive video-microscopy recordings of actual cellular motion (e.g., SI8000 from SONY) can be used to alternatively assess their contractility, however, this method does not directly measure sarcomere motion or force generation kinetics14.
To directly measure sarcomere motion in PSC-CMs, new approaches, such as fluorescent-tagging to sarcomere protein, are emerging. For example, Lifeact is used to label filamentous actin (F-actin) to measure sarcomere motion15,16. Genetically modified iPS cells are another option for tagging sarcomere proteins (e.g., &#945;-actinin [ACTN2] and Myomesin-2 [MYOM2]) by fluorescent protein17,18,19.
In this paper, we describe how to perform time-lapse imaging for measuring sarcomere shortening using Myom2-TagRFP (mouse embryonic stem [ES] cells) and ACTN2-mCherry (human iPS cells). We also show that a patterned culture facilitates sarcomere alignment. In addition, we describe an alternative method of sarcomere labeling, using adeno-associated viruses (AAVs), which can be widely applied to patient-derived iPS cells.

<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.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

Measuring sarcomere shortening using knock-in PSC-CMs reporter lines. Sarcomere-labeled PSC-CMs were used to measure sarcomere shortening. The lines express Myom2-RFP and ACTN2-mCherry from endogenous loci. TagRFP was inserted to Myom2, coding M-proteins that localize to the M-line, while mCherry was knocked-in to ACTN2, coding &#945;-Actinin, which localizes to the Z-line18,25. Time-lapse images...

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.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

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

Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...