Name: isolation, characterization, and differentiation of cardiac stem cells from the adult mouse heart
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart at JoVE.com

This work is supported, in parts, by the National Institutes of Health grants HL-113281 and HL116205 to Paras Kumar Mishra.

The housing, anesthesia, and sacrifice of mice were performed following the approved IACUC protocol of the University of Nebraska Medical Center.
1. Materials
<ol>
	<li>Use 10- to 12-week-old C57BL/6J black male mice, kept in-house at the institutional animal facility, for the isolation of CSCs. CSCs can also be isolated from non-pregnant female mice.</li>
	<li>Sterilize all the necessary surgical instruments, including surgical scissors, fine surgical scissors, curved shank forceps, and a s...

<ol>
	<li>Benjamin, E. 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=Heart+Disease+and+Stroke+Statistics-2017+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association.</a> Circulation. 135, e146 (2017).</li><li>Nguyen, P. K., Rhee, J. W., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Stem+Cell+Therapy+and+Heart+Failure,+2000+to+2016:+A+Systematic+Review.">Adult Stem Cell Therapy and Heart Failure, 2000 to 2016: A Systematic Review.</a> The Journal of the American Medical Association Cardiology. 1, 831-841 (2000).</li><li>Emmert, M. 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=Safety+and+efficacy+of+cardiopoietic+stem+cells+in+the+treatment+of+post-infarction+left-ventricular+dysfunction+-+From+cardioprotection+to+functional+repair+in+a+translational+pig+infarction+model.">Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction - From cardioprotection to functional repair in a translational pig infarction model.</a> Biomaterials. 122, 48-62 (2017).</li><li>Silvestre, J. S., Menasche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Evolution+of+the+Stem+Cell+Theory+for+Heart+Failure.">The Evolution of the Stem Cell Theory for Heart Failure.</a> EBioMedicine. 2, 1871-1879 (2015).</li><li>Terzic, A., Behfar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+for+heart+failure:+Ensuring+regenerative+proficiency.">Stem cell therapy for heart failure: Ensuring regenerative proficiency.</a> Trends in Cardiovascular Medicine. 26, 395-404 (2016).</li><li>Yamada, 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=Embryonic+stem+cell+therapy+of+heart+failure+in+genetic+cardiomyopathy.">Embryonic stem cell therapy of heart failure in genetic cardiomyopathy.</a> Stem Cells. 26, 2644-2653 (2008).</li><li>Sadek, H. A., Martin, C. M., Latif, S. S., Garry, M. G., Garry, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-marrow-derived+side+population+cells+for+myocardial+regeneration.">Bone-marrow-derived side population cells for myocardial regeneration.</a> Journal of Cardiovascular Translational Research. 2, 173-181 (2009).</li><li>Vrtovec, 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=Effects+of+intracoronary+CD34++stem+cell+transplantation+in+nonischemic+dilated+cardiomyopathy+patients:+5-year+follow-up.">Effects of intracoronary CD34+ stem cell transplantation in nonischemic dilated cardiomyopathy patients: 5-year follow-up.</a> Circulation Research. 112, 165-173 (2013).</li><li>Hare, J. 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=Comparison+of+allogeneic+vs+autologous+bone+marrow-derived+mesenchymal+stem+cells+delivered+by+transendocardial+injection+in+patients+with+ischemic+cardiomyopathy:+the+POSEIDON+randomized+trial.">Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial.</a> The Journal of American Medical Association. 308, 2369-2379 (2012).</li><li>Guijarro, D., 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=Intramyocardial+transplantation+of+mesenchymal+stromal+cells+for+chonic+myocardial+ischemia+and+impaired+left+ventricular+function:+Results+of+the+MESAMI+1+pilot+trial.">Intramyocardial transplantation of mesenchymal stromal cells for chonic myocardial ischemia and impaired left ventricular function: Results of the MESAMI 1 pilot trial.</a> International Journal of Cardiology. 209, 258-265 (2016).</li><li>Bobi, 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=Intracoronary+Administration+of+Allogeneic+Adipose+Tissue-Derived+Mesenchymal+Stem+Cells+Improves+Myocardial+Perfusion+But+Not+Left+Ventricle+Function,+in+a+Translational+Model+of+Acute+Myocardial+Infarction.">Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction.</a> Journal of the American Heart Association. 6, (2017).</li><li>Suzuki, E., Fujita, D., Takahashi, M., Oba, S., Nishimatsu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+stem+cells+as+a+therapeutic+tool+for+cardiovascular+disease.">Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease.</a> World Journal of Cardiology. 7, 454-465 (2015).</li><li>Gao, L. R., 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=Intracoronary+infusion+of+Wharton's+jelly-derived+mesenchymal+stem+cells+in+acute+myocardial+infarction:+double-blind,+randomized+controlled+trial.">Intracoronary infusion of Wharton's jelly-derived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial.</a> BMC Medicine. 13, 162 (2015).</li><li>Simpson, D. L., 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+strong+regenerative+ability+of+cardiac+stem+cells+derived+from+neonatal+hearts.">A strong regenerative ability of cardiac stem cells derived from neonatal hearts.</a> Circulation. , S46-S53 (2012).</li><li>Kazakov, 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=C-kit(+)+resident+cardiac+stem+cells+improve+left+ventricular+fibrosis+in+pressure+overload.">C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload.</a> Stem Cell Research. 15, 700-711 (2015).</li><li>Ong, S. 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=Cross+talk+of+combined+gene+and+cell+therapy+in+ischemic+heart+disease:+role+of+exosomal+microRNA+transfer.">Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.</a> Circulation. 130, S60-S69 (2014).</li><li>Sahoo, S., Losordo, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cardiac+repair+after+myocardial+infarction.">Exosomes and cardiac repair after myocardial infarction.</a> Circulation Research. 114, 333-344 (2014).</li><li>Zhang, Z., 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=Pretreatment+of+Cardiac+Stem+Cells+With+Exosomes+Derived+From+Mesenchymal+Stem+Cells+Enhances+Myocardial+Repair.">Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair.</a> Journal of the American Heart Association. 5, (2016).</li><li>Ibrahim, A. G., Cheng, K., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+as+critical+agents+of+cardiac+regeneration+triggered+by+cell+therapy.">Exosomes as critical agents of cardiac regeneration triggered by cell therapy.</a> Stem Cell Reports. 2, 606-619 (2014).</li><li>Emanueli, C., Shearn, A. I., Angelini, G. D., Sahoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+exosomal+miRNAs+in+cardiovascular+protection+and+repair.">Exosomes and exosomal miRNAs in cardiovascular protection and repair.</a> Vascular Pharmacology. 71, 24-30 (2015).</li><li>Menasche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+therapy:+lessons+from+clinical+trials.">Cardiac cell therapy: lessons from clinical trials.</a> Journal of Molecular and Cellular Cardiology. 50, 258-265 (2011).</li><li>Trounson, A., McDonald, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapies+in+Clinical+Trials:+Progress+and+Challenges.">Stem Cell Therapies in Clinical Trials: Progress and Challenges.</a> Cell Stem Cell. 17, 11-22 (2015).</li><li>Takamiya, M., Haider, K. H., Ashaf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+multipotent+sub-population+of+Sca-1(+)+cardiac+progenitor+cells+for+myocardial+regeneration.">Identification and characterization of a novel multipotent sub-population of Sca-1(+) cardiac progenitor cells for myocardial regeneration.</a> PLoS One. 6, e25265 (2011).</li><li>Cambria, E., 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=Translational+cardiac+stem+cell+therapy:+advancing+from+first-generation+to+next-generation+cell+types.">Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types.</a> NPJ Regenerative Medicine. 2, 17 (2017).</li><li>Bruyneel, A. A., Sehgal, A., Malandraki-Miller, S., Carr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapy+for+the+Heart:+Blind+Alley+or+Magic+Bullet?.">Stem Cell Therapy for the Heart: Blind Alley or Magic Bullet?.</a> Journal of Cardiovascular Translational Research. 9, 405-418 (2016).</li><li>Garbern, J. C., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+stem+cell+therapy+and+the+promise+of+heart+regeneration.">Cardiac stem cell therapy and the promise of heart regeneration.</a> Cell Stem Cell. 12, 689-698 (2013).</li><li>Oh, H., Ito, H., Sano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+to+success+in+heart+failure:+Cardiac+cell+therapies+in+patients+with+heart+diseases.">Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases.</a> Journal of Cardiology. 68, 361-367 (2016).</li><li>Smith, 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=Isolation+and+characterization+of+resident+endogenous+c-Kit++cardiac+stem+cells+from+the+adult+mouse+and+rat+heart.">Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.</a> Nature Protocols. 9, 1662-1681 (2014).</li><li>Rutering, 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=Improved+Method+for+Isolation+of+Neonatal+Rat+Cardiomyocytes+with+Increased+Yield+of+C-Kit++Cardiac+Progenitor+Cells.">Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.</a> Journal of Stem Cell Research and Therapy. 5, 1-8 (2015).</li><li>Saravanakumar, M., Devaraj, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+homing+pattern+of+c-kit++Sca-1++CXCR4++resident+cardiac+stem+cells+in+neonatal,+postnatal,+and+adult+mouse+heart.">Distribution and homing pattern of c-kit+ Sca-1+ CXCR4+ resident cardiac stem cells in neonatal, postnatal, and adult mouse heart.</a> Cardiovascular Pathology. 22, 257-263 (2013).</li><li>Monsanto, M. 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=Concurrent+Isolation+of+3+Distinct+Cardiac+Stem+Cell+Populations+From+a+Single+Human+Heart+Biopsy.">Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.</a> Circulation Research. 121, 113-124 (2017).</li><li>Vidyasekar, P., Shyamsunder, P., Santhakumar, R., Arun, R., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+protocol+for+the+isolation+and+culture+of+cardiomyocytes+and+progenitor+cells+from+neonatal+mouse+ventricles.">A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles.</a> European Journal of Cell Biology. 94, 444-452 (2015).</li><li>Dergilev, K. V., 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=Comparison+of+cardiac+stem+cell+sheets+detached+by+Versene+solution+and+from+thermoresponsive+dishes+reveals+similar+properties+of+constructs.">Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs.</a> Tissue Cell. 49, 64-71 (2017).</li><li>Zaruba, M. M., Soonpaa, M., Reuter, S., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyogenic+potential+of+C-kit(+)-expressing+cells+derived+from+neonatal+and+adult+mouse+hearts.">Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts.</a> Circulation. 121, 1992-2000 (1992).</li><li>Wang, 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=Isolation+and+characterization+of+a+Sca-1+/CD31-+progenitor+cell+lineage+derived+from+mouse+heart+tissue.">Isolation and characterization of a Sca-1+/CD31- progenitor cell lineage derived from mouse heart tissue.</a> BMC Biotechnology. 14, 75 (2014).</li><li>Smits, A. 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=Human+cardiomyocyte+progenitor+cells+differentiate+into+functional+mature+cardiomyocytes:+an+in+vitro+model+for+studying+human+cardiac+physiology+and+pathophysiology.">Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology.</a> Nature Protocols. 4, 232-243 (2009).</li></ol>

The critical steps of this CSC isolation protocol are as follows. 1) A sterilized condition must be maintained for extraction of the hearts from the mice. Any contamination during the heart extraction may compromise the quality of the CSCs. 2) The blood must be completely removed before mincing the heart, which is done by several washes of the whole heart and the heart pieces with HBSS solution. 3) The heart pieces must be completely lysed into a single-cell suspension with collagenase solution. 4) The polysucrose and so...

Ischemic heart disease, including myocardial infarction (MI), is a major cause of death around the world1. Stem cell therapy for regenerating dead myocardium remains a major approach to improve the cardiac function of an MI heart2,3,4,5. Different types of stem cells have been used to replenish dead myocardium and to improve the cardiac function of an MI heart. They can be broadly categorized into embryonic stem cells6 and adult stem cells. In adult stem cells, various types of stem cells have been used, such as bone marrow-derived mononuclear cells7,8, mesenchymal stem cells derived from bone marrow9,10, adipose tissue11,12, and umbilical cord13, and CSCs14,15. Stem cells can promote cardiac regeneration through endocrine and/or paracrine actions16,17,18,19,20. However, a major limitation of stem cell therapy is obtaining an adequate number of stem cells that can proliferate and/or differentiate toward a specific cardiac lineage21,22. Autologous and allogenic transplantation of stem cells is an important challenge in stem cell therapy9. CSCs could be a better approach for cardiac regeneration because they are derived from the heart and they can be more easily differentiated into cardiac lineages than non-cardiac stem cells. Thus, it reduces the risk of teratoma. In addition, the endocrine and paracrine effects of CSCs, such as exosomes and miRNAs derived from the CSCs, could be more effective than other types of stem cells. Thus, CSCs remains a better option for cardiac regeneration23,24.
Although CSCs are a better candidate for cardiac regeneration in an MI heart due to their cardiac origin, a major limitation with CSCs is less yield due to the lack of an efficient isolation method. Another limitation could be the impaired differentiation of CSCs toward cardiomyocytes lineage2,25,26,27. To circumvent these limitations, it is important to develop an efficient protocol for CSC isolation, characterization, and differentiation towards cardiac lineage. There is no single acceptable marker for CSCs and a specific cell-surface marker-based isolation of CSCs yields less CSCs. Here, we standardize a simple gradient centrifugation approach to isolate CSCs from the mouse heart that is cost-effective and results in an increased yield of CSCs. These isolated CSCs can be selected for specific cell-surface markers by fluorescence-activated cell shorting. In addition to CSCs isolation, we provided a protocol for CSC culture, characterization, and differentiation towards cardiomyocyte lineage. Thus, we present an elegant method to isolate, characterize, culture, and differentiate CSCs from adult mouse hearts (Figure 5).

<table>
	<tbody>
		<tr>
			<td>Mice</td>
			<td>The Jackson laboratory, USA</td>
			<td>Stock no. 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4-</td>
			<td>Abcam</td>
			<td>ab18976 (rabbit polyclonal)</td>
			<td>OCT4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Abcam</td>
			<td>ab97959 (rabbit polyclonal)</td>
			<td>SOX2-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Nanog</td>
			<td>Abcam</td>
			<td>ab80892 (rabbit polyclonal)</td>
			<td>Nanog-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Abcam</td>
			<td>ab16667 (rabbit polyclonal)</td>
			<td>Ki67-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Sca I</td>
			<td>Millipore</td>
			<td>AB4336 (rabbit polyclonal)</td>
			<td>Sca I Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>NKX2-5</td>
			<td>Santa Cruz</td>
			<td>sc-8697 (goat polyclonal)</td>
			<td>NKX2-5-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>GATA4</td>
			<td>Abcam</td>
			<td>ab84593 (rabbit polyclonal)</td>
			<td>GATA4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>MEF2C</td>
			<td>Santa Cruz</td>
			<td>sc-13268 (goat polyclonal)</td>
			<td>MEF2C-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Troponin I</td>
			<td>Millipore</td>
			<td>MAB1691 (mouse monoclonal)</td>
			<td>Troponin I-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Actinin</td>
			<td>Millipore</td>
			<td>MAB1682 (mouse monoclonal)</td>
			<td>Actinin-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>ANP</td>
			<td>Millipore</td>
			<td>AB5490 (mouse polyclonal)</td>
			<td>ANP-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Alex Fluor-488 checken anti-rabbit</td>
			<td>Life technology</td>
			<td>Ref no. A21441</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 goat anti-rabbit</td>
			<td>Life technology</td>
			<td>Ref no. A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 rabbit anti-goat</td>
			<td>Life technology</td>
			<td>Ref no. A11078</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-488 checken anti-mouse</td>
			<td>Life technology</td>
			<td>Ref no. A21200</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 checken anti-goat</td>
			<td>Life technology</td>
			<td>Ref no. A21468</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture medium: </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CSC maintenance medium</td>
			<td>Millipore</td>
			<td>SCM101</td>
			<td>Note: For CSC culture, PBS or incomplete DMEM medium was used for washing the cells</td>
		</tr>
		<tr>
			<td>cardiomyocytes differentiation medium</td>
			<td>Millipore</td>
			<td>SCM102</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell Isolation buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polysucrose and sodium diatrizoate solution (Histopaque1077)</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>2018-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase solution</td>
			<td>STEMCELL Technologies</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>LONZA</td>
			<td>S1226</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermoscientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Other reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal checken serum</td>
			<td>Vector laboratory</td>
			<td>S3000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Applichem</td>
			<td>A100,0010</td>
			<td>Dapi, working concentration-1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Biorad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ACROS</td>
			<td>Cas No. 900-293-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Sceintific</td>
			<td>Lot No. 160170</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue culture materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flask</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell stainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell stainer</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>09-719C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syring</td>
			<td>BD</td>
			<td>Ref no. 309604</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L, 200 &#181;L, 1000 &#181;L pipette tips</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL, 10mL, 25 mL disposible plastic pipette</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrufuge machine</td>
			<td>Thermo Scientific</td>
			<td>LEGEND X1R centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS microscope</td>
			<td>Life technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting slide</td>
			<td>Biorad</td>
			<td>145-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippte aid</td>
			<td>Thermo Scientific</td>
			<td>S1 pipet filler</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Instruments:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine surgical scissors</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curve shank forceps</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, characterization, and differentiation of cardiac stem cells from the adult mouse heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

In the present study, we isolated CSCs from 10- to 12-week-old C57BL/6J male mice hearts. Here, we have presented a simple method for CSC isolation and characterization using markers of pluripotency. We also presented an elegant method for CSC differentiation and the characterization of CSCs that differentiated toward cardiomyocytes lineage. We observed a spindle shape morphology of 2- to 3-days-cultured CSCs under a phase-contrast microscope (Figure&#160;1A ...

Watch this Scientific Journal Video about Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart at JoVE.com

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...