Name: generation of human cardiomyocytes: a differentiation protocol from feeder-free human induced pluripotent stem cells
Uploaded: 2013-06-28T14:00:00
Description: Watch this Scientific Journal Video about Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells at JoVE.com

BS was supported by University of Milan-Bicocca Summer Student Research Training Program; research was supported from funds of the Italian Ministry of Health and Italian Ministry of Education, University and Research and Fondazione Humanitas to GC; we thanks Michael V.G. Latronico for critically reading the manuscript.

1. Feeder-free Maintenance and Passaging of Human iPS Cell Lines
<ol>
	<li>Prepare the Matrigel-coated dishes. Thaw one vial of human ESC-qualified Matrix on ice for one hour and dilute it in 25 ml DMEM-F12 medium. Add 1 ml to each 35 mm plate (or equivalent amounts per surface area if other dishes are used), keeping everything on ice and ensuring that all plates and tubes are pre-cooled. Cover plates with aluminum foil.</li>
</ol>
Note: Matrigel stock concentrations vary by the...

<ol>
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R., Gelb, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+cardiomyocytes+as+models+for+genetic+cardiovascular+disorders.">Induced pluripotent stem cell-derived cardiomyocytes as models for genetic cardiovascular disorders.</a> Curr. Opin. Cardiol. 26, 223-229 (2011).</li><li>Park, I. 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=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</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, 808-812 (2012).</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> N. Engl. J. Med. 363, 1397-1409 (2010).</li><li>Yazawa, 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=Using+induced+pluripotent+stem+cells+to+investigate+cardiac+phenotypes+in+Timothy+syndrome.">Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome.</a> Nature. 471, 230-234 (2011).</li><li>Itzhaki, 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=Modeling+of+catecholaminergic+polymorphic+ventricular+tachycardia+with+patient-specific+human-induced+pluripotent+stem+cells.">Modeling of catecholaminergic polymorphic ventricular tachycardia with patient-specific human-induced pluripotent stem cells.</a> Journal of the American College of Cardiology. 60, 990-1000 (2012).</li><li>Jung, C. 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=Dantrolene+rescues+arrhythmogenic+RYR2+defect+in+a+patient-specific+stem+cell+model+of+catecholaminergic+polymorphic+ventricular+tachycardia.">Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.</a> EMBO Molecular Medicine. 4, 180-191 (2012).</li><li>Sun, 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=Patient-specific+induced+pluripotent+stem+cells+as+a+model+for+familial+dilated+cardiomyopathy.">Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.</a> Science Translational Medicine. 4, 130ra147 (2012).</li><li>Kehat, 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=Human+embryonic+stem+cells+can+differentiate+into+myocytes+with+structural+and+functional+properties+of+cardiomyocytes.">Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.</a> The Journal of Clinical Investigation. 108, 407-414 (2001).</li><li>Cao, 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=Ascorbic+acid+enhances+the+cardiac+differentiation+of+induced+pluripotent+stem+cells+through+promoting+the+proliferation+of+cardiac+progenitor+cells.">Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells.</a> Cell Research. 22, 219-236 (2012).</li><li>Takahashi, T., 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=Ascorbic+acid+enhances+differentiation+of+embryonic+stem+cells+into+cardiac+myocytes.">Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.</a> Circulation. 107, 1912-1916 (2003).</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> N. Engl. J. Med. 363, 1397-1409 (2011).</li><li>Watanabe, K., 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+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25, 681-686 (2007).</li></ol>

Pluripotent stem cells have the potential to differentiate spontaneously into CMs, albeit with low efficiency and high variability among lines. The development of novel induction methods has therefore focused on improving the efficiency of the process and moving toward more defined protocols. However, most of these new differentiation methods are quite complex, requiring elaborate culture conditions, fine control of timing and concentration of reagents, and treatment with expensive cytokines and growth factors. Also, dif...

Historically, the investigation of the genetic and molecular mechanisms driving human development and disease has been based on the generation of genetically modified animal models. However, numerous human phenotypes fail to be successfully replicated in mice, mainly because of the biological differences existing between the two species. On the other hand, access to human tissues may be limited and often do not allow enough material to be obtained for in-depth experimental studies. The field of cardiovascular biology suffers from both these limitations: the physiology of the human heart is significantly different from that of the mouse, and a significant quantity of heart tissue is accessible only post-mortem or during heart surgery. Finding an optimal source for the differentiation of functional human CMs has therefore become a central topic in cardiovascular biology, and much effort has been made to address this issue. Various cells types have been proposed, including skeletal myoblasts, local cardiac stem cells, bone marrow mononuclear cells, endothelial progenitors, and mesenchymal stem cells. However, data obtained using these cells have not been consistent 4.
The derivation of human embryonic stem cells (ESC) first 5 and the groundbreaking discovery of induced pluripotent stem (iPS) cells by Yamanaka and Thomson 6,7 later seemed to provide solutions: these cells have the ability to grow indefinitely and the potential to give rise to all cell derivatives of the three germ layers, including the CMs. The use of iPS cells offers further advantages: being derived from autologous adult cells, they carry the same genome as the individual or patient from which they are derived. They therefore allow the development of in vitro models that facilitate the investigation of human genetic diseases and mechanisms of development. In virtue of this characteristic, iPS cells can also overcome problems related to immune rejection and ethical issues 8. For these reasons, even though ESCs still represent the gold standard in the field of stem cell biology, researchers worldwide are now moving more toward iPS cell technology.
iPS cells are now being employed to model human disease in vitro for various conditions, including congenital cardiovascular disorders 9,10. Recently, application of iPS cell disease modeling has been performed for monogenic cardiac disorders (i.e. long QT syndromes, catecholaminergic polymorphic ventricular tachycardia) and pathologies in which cardiac defects are part of a complex phenotype (i.e. Leopard and Timothy syndromes, dilated cardiomyopathy). These reports confirmed that patient-specific iPS cells that are differentiated into CMs display similar phenotypic and functional characteristics as the disease in vivo.
However, there are still many challenges to improve the efficacy of inducing the iPS cells into the cardiac lineage. Spontaneous generation of CMs from human ESCs via aggregate formation called embryoid bodies (EBs) have proven successful 17. Since this discovery, many other methods have been proposed. Many groups have greatly enhanced the efficiency of the differentiation protocol and have moved toward chemically defined and animal-product-free culture reagents (see Mummery, C., et al., Circulation Research (2012) for a comprehensive review of all existing methods 3).
Nevertheless, the classical method based on EB aggregation still represents the most commonly employed for performing functional studies and investigating disease mechanisms. Our proposed protocol is based on the aggregation of iPS cells into EBs and culture in the presence of serum and ascorbic acid, which has been shown to enhance the cardiac differentiation process and to positively impact the maturation of these cells 18,19. In this article we will go through this methodology in detail and will show how to use feeder-free iPS cell lines for generating patient-specific iPS-derived CMs.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media and Reagents</td>
		</tr>
		<tr>
			<td>Human ESC-qualified Matrix</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Thaw the 5 ml bottle at 4 &deg;C on ice O/N, aliquot in pre-chilled cryo-tubes on ice, according to the data sheet dilution instructions and store at -20 &deg;C .</td>
		</tr>
		<tr>
			<td>Fibronectin, from human plasma, 0.1% solution</td>
			<td>Sigma</td>
			<td>F0896-2MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>PBS (no Mg and Ca), 10X</td>
			<td>Lonza</td>
			<td>BE17-515F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (with Mg and Ca), 10X</td>
			<td>Bio Sera</td>
			<td>XC-S2067/500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II, neutral protease, grade II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>Stock: 10 mg/ml in DMEM-F12 at -20 &deg;C, working solution: 1 mg/ml in PBS (no Mg/Ca)</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA, 0.5%</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase type 2</td>
			<td>Worthington</td>
			<td>4176</td>
			<td>Dilute to 480 U/ml (e.g. 1.6 mg/ml) in PBS with Mg and Ca</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>21331-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Thaw at 4 &deg;C, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (origin South America)</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>Batches should be tested for their cardiogenic potential. Thaw at 4 &deg;C, heat-inactivate at 56 &deg;C for 30 min, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>PenStrep, penicillin-Streptomycin, 100X</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GlutaMAX, 100X</td>
			<td>Life Technologies</td>
			<td>35050-038</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM NEAA, 100X</td>
			<td>Invitrogen</td>
			<td>11140-135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 supplement, 100X</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 supplement, 50X</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin, from porcine skin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td>Make stock at 1% in water, store at -20 &deg;C. Dilute to 0.1% in PBS and keep at 4 &deg;C</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td>Combine Supplement 5X with the basic medium, aliquot and store at -20 &deg;C. Do not keep complete medium at 4 &deg;C for more than 1 week</td>
		</tr>
		<tr>
			<td>Nutristem hESC XF</td>
			<td>Stemgent from Biological Industries</td>
			<td>05-100-1A</td>
			<td>Thaw at 4 &deg;C O/N, aliquot and store at -20 &deg;C until use. Aliquots may be kept at 4 &deg;C for no more than a week.</td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>Stem Cell Technologies</td>
			<td>5853</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-25G</td>
			<td>Prepare stocks at 10 mg/ml in water. Store aliquots at -20 &deg;C in the dark. Add to EBM at the final concentration (500X)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>35 mm culture dishes</td>
			<td>Becton Dickinson</td>
			<td>353001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Becton Dickinson</td>
			<td>353043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permanox 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177437</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-well plates, ultra-low-attachment surface</td>
			<td>Corning Incorporated</td>
			<td>3471</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Becton Dickinson</td>
			<td>304622</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes, 230 mm</td>
			<td>VWR International</td>
			<td>612-1702</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Becton Dickinson</td>
			<td>300188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipments</td>
		</tr>
		<tr>
			<td>IVF Workstation</td>
			<td>KSystem, by Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nikon SMZ1500 Stereomicroscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

In our studies we generated CMs from several iPS cell lines. These lines were initially generated on an MEF feeder layer but were immediately placed on Matrigel using a defined medium (either mTESR1 or Nutristem). Various assays were used to verify maintenance of the morphological properties, the expression of markers typical of pluripotent cells (OCT-4, SSEA4 and TRA1-60) and their genome stability (Figure 1).
Induction into the cardiac lineage was triggered by aggregation of...

Watch this Scientific Journal Video about Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells at JoVE.com

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

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

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