We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

1. Derivation of Mouse Embryonic CPCs from Mouse ESCs
<ol>
	<li>Prepare mouse embryonic fibroblasts (MEFs) feeder layer.
	<ol>
		<li>Warm MEF medium (10% FBS in DMEM) to 37 &#176;C.</li>
		<li>Prepare gelatin coated plates.
		<ol>
			<li>Add 1 ml of 0.1% Gelatin in water into a well of 6-well plate or 5 ml into a 10 cm dish.</li>
			<li>Leave plates or dishes at 37 &#176;C or room temperature for at least 30 min. Aspire the gelatin prior to use.</li>
		</ol>
		</li>
		<li>Thaw irradiated MEFs quickly in a 37 &#176;C water bath, with gentle shaking.</li>
		<li>Transfer the cells gently to a 15 ml or 50 ml conical tube. Add 5 ml of pre-warmed MEF medium. Swirl the cells slowly and centrifuge at 200 x g for 5 min.</li>
		<li>Resuspend cells in MEF medium and count viable cells. Use the following volumes of MEF medium: 2 ml for cells per well from a 6-well plate and 10 ml for cells from a 10 cm dish.</li>
		<li>Plate cells into 6-well plates or 10 cm dishes at 1-2 x 106 per plate/dish.</li>
		<li>Incubate the MEF plates/dishes at 37 &#176;C, 5% CO2 at least 24 hr prior to use. 
		NOTE: Discard MEF plates/dishes older than a week.</li>
	</ol>
	</li>
	<li>Prepare mouse ESCs
	<ol>
		<li>Culture the Isl1-cre; Rosa26YFP/ Mef2c-cre; Rosa26YFP mouse ESCs on MEFs till 90% confluent. Use ES cell medium comprised of 410 ml DMEM, 75 ml FBS, 0.1 mM MEM NEAA, 2 mM L-glutamine, 0.1 mM Pyruvate, 100 U Pen/Strep, and 0.1 mM &#946;-mercaptoethanol.</li>
		<li>Prepare gelatin coated 6-well plates as described in 1.1.2.</li>
		<li>Aspirate medium from plates and rinse cells with DPBS.
		<ol>
			<li>Aspirate DPBS and add 0.4 ml 0.25% Trypsin into one well of 6-well plate. Incubate cells at 37 &#176;C, 5% CO2 for 5 min. Add 1 ml pre-warmed ES medium to cease Trypsin reaction.</li>
		</ol>
		</li>
		<li>Harvest cells and centrifuge cells at 200 x g and aspirate medium. Resuspend cells in 1 ml ES cell medium and count the cells.</li>
		<li>Plate ESCs on gelatin-coated plates at density of 3 x 104/cm2. Incubate at 37 &#176;C, 5% CO2 for about 2 days when ESCs reach about 90% confluent. Change the medium everyday.</li>
		<li>When ESCs are 90% confluent, repeat Steps 1.2.2-1.2.5 to completely remove the MEF feeders.</li>
	</ol>
	</li>
	<li>Induce CPC differentiation by EB formation
	<ol>
		<li>Prepare differentiation medium as followed: 36 ml IMDM, 12 ml Ham&#8217;s F12, 2 mM L-glutamine, 0.34 ml BSA (7.5%), 0.25 ml N2, 0.5 ml B27, 50 &#181;g/ml AA, and 0.45 mM 1-thioglycerol.</li>
		<li>Aspirate medium from cells and rinse with DPBS. Aspirate DPBS and add 0.4 ml 0.25% Trypsin into one well of 6-well plate. Incubate at 37 &#176;C for 5 min.</li>
		<li>Add 1 ml differentiation medium to cease the reaction and pipette up and down to disperse cell clusters into single cells.</li>
		<li>Centrifuge ESCs at 200 x g for 5 min and discard the medium.
		<ol>
			<li>Resuspend 1 x 106 ESCs in 1 ml differentiation medium. Count the cells, and culture the cells in low detachment Petri dish at concentration of 1 x 105 cells/ml in differentiation medium at 37 &#176;C for first EB aggregation.</li>
		</ol>
		</li>
		<li>Two days after EB formation, transfer EBs from dishes to 50 ml tubes. Spin at 200 x g for 1 min. Remove medium and rinse EBs with 10 ml DPBS without Ca2+ and Mg2+.</li>
		<li>Aspirate DPBS and add 1 ml 0.25% Trypsin. Incubate at 37 &#176;C for 5 min. Add 4.5 ml differentiation medium to stop the reaction. Pipet up and down to dissociate EBs into single cells.</li>
		<li>Centrifuge the cells at 200 x g for 5 min. Aspirate medium and resuspend cells in 1 ml differentiation medium.</li>
		<li>Count the cells, and dilute 2 x 106 cells to 1 x 105 cells/ml in differentiation medium. Add VEGF, Bmp4 and Activin A to the medium at final concentration of 5 ng/ml, 0.8 ng/ml, and 5 ng/ml, respectively. Culture the cells in Petri dish for second EB formation at 37 &#176;C.</li>
		<li>40 hr after second EB formation, transfer EBs to 50 ml tubes. Rinse with DPBS once, dissociate EBs with 1 ml 0.25% Trypsin at 37 &#176;C for 5 min. Pipet up and down to dissociate EBs into single cells.</li>
		<li>Resuspend the cells in Stempro-34 medium with 5 ng/ml VEGF, 10 ng/ml bFGF and 12.5 ng/ml FGF10. Plate the cells at 2 x 105/cm2 in gelatin-coated plates. Culture in 37 &#176;C incubator for about 32 hr before CPC isolation.</li>
	</ol>
	</li>
	<li>Isolate mCPCs
	<ol>
		<li>Aspirate medium and rinse with DPBS. Add 0.5 ml 0.25% Trypsin to culture plates at 37 &#176;C for 5 min. Add 4.5 ml differentiation medium to stop the reaction. Pipet up and down to dissociate EBs into single cells. Collect the cells by centrifuge and resuspend cells in 2% FBS/PBS for FACS-purification.</li>
		<li>Run undifferentiated ESCs on sorter as the negative control. Change voltage to adjust forward scatter (FSC) and side scatter (SSC) to select desired population. Use forward scatter versus side scatter and forward scatter height versus width to exclude debris and doublet. In the selected sub-population, according to the distribution of ESC controls, draw a gate of YFP positive on histogram to eliminate cell autofluorescence. Collect YFP+ CPCs from YFP+ gating.</li>
		<li>Plate the purified CPCs (YFP+ cells) in 6-well plates with differentiation medium (1.3.1). Culture additional 3-5 days at 37 &#176;C for differentiation of CPCs into cardiomyocytes and SMCs.</li>
	</ol>
	</li>
</ol>
2. Derivation of Human Embryonic CPCs from Human ESCs
<ol>
	<li>Prepare Feeders
	<ol>
		<li>Prepare mouse embryonic fibroblasts (MEFs) feeder layer as described above at a density of 2 x 104/cm2.</li>
	</ol>
	</li>
	<li>Maintain human ESCs
	<ol>
		<li>Maintain human ISL1:cre;pCAG-flox-STOP-flox-GFP or RFP ESCs routinely on MEF using regular hESC medium (Knockout DMEM/F-12 385 ml, Knockout SR 100 ml, 2 mM L-glutamine, 0.1 mM NEAA, 0.1 mM &#946;-mercaptoethanol, 10 ng/ml basic FGF). Prior to cardiac differentiation, transfer human ESCs in feeder free condition for at least 2 passages.</li>
	</ol>
	</li>
	<li>Prepare human ESCs in feeder free condition
	<ol>
		<li>Prepare coated plates for human ESCs culture.
		<ol>
			<li>Thaw matrigel at 4 &#176;C overnight. Put a 50 ml tube on ice and add 30 ml cold DMEM/F12 medium into the tube.</li>
			<li>Add 1 ml matrigel into the cold medium and mix well. Add 1 ml cold matrigel-DMEM/12 medium into a well of 6-well plate or 5 ml into a 10 cm dish.</li>
			<li>Leave plates/dishes at 4 &#176;C overnight or room temperature for 2 hr. Aspirate medium prior to use.</li>
		</ol>
		</li>
		<li>Split human ESCs onto the plates: Aspirate media from MEF plate and rinse human ESCs with DPBS. Then add 1 ml dispase (1 mg/ml) to a well of 6-well plate. Incubate cells in 37 &#176;C incubator until edges of colonies start to detach.</li>
		<li>Remove dispase solution. Rinse with DPBS twice, then add 2.5 ml/per well mTeSR medium or MEF-conditioned medium to the plate.</li>
		<li>Scrape cells using a cell scraper and carefully pipet up and down to break human ESC colonies into small clumps.</li>
		<li>Transfer the ESC clumps and dilute 1:3 into the coated plates.</li>
		<li>Add 2 ml/well mTeSR medium or MEF-conditioned medium and change medium daily.</li>
		<li>Culture human ESCs in mTeSR medium or MEF-conditioned medium on coated plates for at least 2 passages.</li>
	</ol>
	</li>
	<li>Induce CPC differentiation from human ESCs
	<ol>
		<li>When cells are ~90% confluent, aspirate the medium and rinse with DPBS. Then incubate the hESCs in 0.5 mg/ml Dispase at 37 &#176;C for 20 min.</li>
		<li>Rinse the hESCs with DPBS twice, remove cells from plates with a cell lifter, then resuspend cell clamps in differentiation medium (DMEM/F12 containing 18% FBS, 0.1 mM NEAA, 2 mM L-glutamine, 0.1 mM &#946;-mercaptoethanol and 50 &#181;g/ml Ascorbic Acid).</li>
		<li>Transfer cells into 6-well ultra-low attachment plates for EB formation.</li>
		<li>Change differentiation medium next day.</li>
		<li>Replace medium every 3 days and maintain EBs in suspension culture.</li>
	</ol>
	</li>
	<li>Isolate hCPCs
	<ol>
		<li>Collect EBs no later than day 9 of differentiation for human CPC isolation.</li>
		<li>Aspirate medium and rinse EBs with DPBS twice. Add 0.5 ml 0.25% Trypsin to each well of 6-well culture plates at 37 &#176;C for 5 min.</li>
		<li>Add equal volume of differentiation medium to stop the reaction. Pipet up and down to dissociate EBs into single cells. Collect the cells by centrifuge at 200 x g for 5 min and resuspend cells in 2% FBS/DPBS. Filter cells with 40 &#181;m cell strainer and FACS-purified the RFP+ CPC cells as described in 1.4.3.</li>
		<li>Plate sorted hCPCs onto geltin-coated plates. Culture hCPCs in differentiation medium for 7-9 days to differentiate into cardiomyocyte and smooth muscle cells. 7 days after plating, culture differentiated cells with 5% Knockout SR in DMEM/F12 medium.</li>
	</ol>
	</li>
</ol>

<ol>
	<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 (6), 689-698 (2013).</li><li>Passier, R., van Laake, L. W., Mummery, C. L. <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-based+therapy+and+lessons+from+the+heart.">Stem-cell-based therapy and lessons from the heart.</a> Nature. 453 (7193), 322-329 (2008).</li><li>Bondue, A., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesp1:+a+key+regulator+of+cardiovascular+lineage+commitment.">Mesp1: a key regulator of cardiovascular lineage commitment.</a> Circulation Research. 107 (12), 1414-1427 (2010).</li><li>Bondue, 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=Mesp1+acts+as+a+master+regulator+of+multipotent+cardiovascular+progenitor+specification.">Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification.</a> Cell Stem Cell. 3 (1), 69-84 (2008).</li><li>Bu, 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=Human+ISL1+heart+progenitors+generate+diverse+multipotent+cardiovascular+cell+lineages.">Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.</a> Nature. 460 (7251), 113-117 (2009).</li><li>Lei, I., Gao, X., Sham, M. H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+protein+component+BAF250a+regulates+cardiac+progenitor+cell+differentiation+by+modulating+chromatin+accessibility+during+second+heart+field+development.">SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development.</a> The Journal of Biological Chemistry. 287 (29), 24255-24262 (2012).</li><li>Lei, I., Liu, L., Sham, M. H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+in+cardiac+progenitor+cell+differentiation.">SWI/SNF in cardiac progenitor cell differentiation.</a> Journal of Cellular Biochemistry. 114 (11), 2437-2445 (2013).</li><li>Wu, S. 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=Developmental+origin+of+a+bipotential+myocardial+and+smooth+muscle+cell+precursor+in+the+mammalian+heart.">Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.</a> Cell. 127 (6), 1137-1150 (2006).</li><li>Spater, 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=A+HCN4++cardiomyogenic+progenitor+derived+from+the+first+heart+field+and+human+pluripotent+stem+cells.">A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells.</a> Nature Cell Biology. 15 (9), 1098-1106 (2013).</li><li>Verzi, M. P., McCulley, D. J., De Val, S., Dodou, E., Black, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+ventricle,+outflow+tract,+and+ventricular+septum+comprise+a+restricted+expression+domain+within+the+secondary/anterior+heart+field.">The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.</a> Developmental Biology. 287 (1), 134-145 (2005).</li><li>Boheler, K. 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=Differentiation+of+pluripotent+embryonic+stem+cells+into+cardiomyocytes.">Differentiation of pluripotent embryonic stem cells into cardiomyocytes.</a> Circulation Research. 91 (3), 189-201 (2002).</li><li>Wada, 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=Induction+of+human+cardiomyocyte-like+cells+from+fibroblasts+by+defined+factors.">Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 12667-12672 (2013).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell Stem Cell. 8 (2), 228-240 (2011).</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 (14), 1912-1916 (2003).</li><li>Wamstad, J. 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=Dynamic+and+coordinated+epigenetic+regulation+of+developmental+transitions+in+the+cardiac+lineage.">Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.</a> Cell. 151 (1), 206-220 (2012).</li><li>Cong, 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Ding, Q., 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=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell Stem Cell. 12 (4), 393-394 (2013).</li><li>Gaj, T., Gersbach, C. S., Barbas, T. A. L. E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Joung, J. K., Sander, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALENs:+a+widely+applicable+technology+for+targeted+genome+editing.">TALENs: a widely applicable technology for targeted genome editing.</a> Nature Reviews. Molecular Cell Biology. 14 (1), 49-55 (2013).</li><li>Yang, 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=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Laflamme, M. 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=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature. biotechnology. 25 (9), 1015-1024 (2007).</li><li>Burridge, P. W., 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+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PloS One. 6 (4), e18293 (2011).</li><li>Kouskoff, V., Lacaud, G., Schwantz, S., Fehling, H. J., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+development+of+hematopoietic+and+cardiac+mesoderm+during+embryonic+stem+cell+differentiation.">Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (37), 13170-13175 (2005).</li><li>Xu, X. Q., 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=Chemically+defined+medium+supporting+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.</a> Differentiation: Research in Biological Diversity. 76 (9), 958-970 (2008).</li><li>Lian, 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-1857 (2012).</li><li>Bizy, 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=Myosin+light+chain+2-based+selection+of+human+iPSC-derived+early+ventricular+cardiac+myocytes.">Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.</a> Stem Cell Research. 11 (3), 1335-1347 (2013).</li></ol>

The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Heart disease remains the leading cause in the world today and the death rates have remained virtually unchanged in the past two decades (American Heart Association). There is a critical need for developing novel therapeutic strategies to effectively prevent or reverse heart failure. One promising strategy is cell-based therapy following the rapid development of stem cell biology1. In this regard, multipotent CPCs could be an excellent cell source for the therapy due to their ability to proliferate but committed only to cardiac lineage differentiation. Therefore, efficient and robust method to generate and isolate CPCs is of great importance for heart cell therapy studies.
This protocol focuses on embryonic CPCs identified during early embryogenesis and how their generation from ESCs. Various CPCs have been isolated from embryonic and adult hearts, even from bone marrows2. During embryo development, bone morphogenetic proteins (BMPs), wingless-type MMTV integration site family members (Wnts) and Nodal signals induce the commitment of the Mesp1+ multipotent mesoderm3. Mesp1+ cells then differentiate into the embryonic CPCs4. These CPCs are typically marked by HCN4, NK2 homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5), and myocyte enhancer factor 2C (Mef2c), form primary and second heart fields, and contribute to the major parts of heart during cardiogenesis5-10. Both Nkx2-5+ and Isl1+/Mef2c+ CPCs are able to differentiate into cardiomyocytes, smooth muscle cells (SMCs), and endothelial cells5-8. Thus these CPCs will give rise to cardiac vasculature as well as cardiac tissues and are an ideal cell source for cell-based heart therapy. Consequently, generating CPCs in vitro has been a major research focus in cardiovascular studies. Since ESCs have unlimited expansion capacity and represent the ICM cells at the blastocyst stage, differentiation of ESCs into embryonic CPCs following the natural embryogenesis is considered a logical and effective approach to obtain CPCs.
One widely applied approach to obtain CPCs from ESCs is to aggregate ESCs into EBs11. To improve the differentiation efficiency, defined chemical and growth factors based on the knowledge of cardiac development have been used12-14. However, there are no definitive CPC markers, particularly no cell-surface markers, which are widely accepted in the field. To address this issue, ESCs are engineered to mark Isl1+ or Mef2c+ CPCs and their derivatives with fluorescent reporters using Cre/loxP system. The cre recombinase is knocked in under the control of Isl1/Mef2c promoter/enhancer. Modified fluorescent protein RFP or YFP gene driven by a constitutive promoter can be activated by excision of flox stop codon with cre recombinase (ISL1:cre;pCAG-flox-STOP-flox-GFP or RFP/Isl1-cre;Rosa26YFP/Mef2c-cre;Rosa26YFP)5,6. Once the ESCs are differentiated into second heart field CPCs, Isl1/Mef2c promoter/enhancer driven cre will activate the fluorescent reporters and CPCs can be enriched by FACS-purification. Briefly, EB aggregation method is used to initiate ESC differentiation. To enhance differentiation efficiency, the differentiated cells are treated with ascorbic acid (AA) and growth factors such as Bmp4, Activin A and Vegf13,15. This protocol allows robust and efficient CPC differentiation using both mouse and human ESCs.

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derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

The protocol shows derivation of CPCs from several ES cell lines. ESCs are aggregated to form EBs to differentiate into CPCs. ESCs are routinely maintained on MEF feeders (Figure 1A, F) and feeders are removed before differentiation. Upon aggregation of ESCs into EBs in differentiation medium (Figure 1B, G), the second formed EBs are treated with Bmp4 and Activin A to enhance the mesoderm differentiation in mouse cell lines (Figure 1C). ESCs are differentiated into cardi...

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Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Research

JoVE Journal

Developmental Biology

8.1K Views.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Video: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biology

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetics

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

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

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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.

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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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