Name: in vitro generation of heart field-specific cardiac progenitor cells
Uploaded: 2019-07-03T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells at JoVE.com

E. T. was supported by The Magic That Matters and AHA. C. K. was supported by grants from NICHD/NIH (R01HD086026), AHA, and MSCRF.&#160;

NOTE: In vitro generation of heart field-specific mouse cardiac progenitor cells (Figure 1).
1. Maintenance of Mouse ESCs
<ol>
	<li>Grow mESCs (mESCsTbx1-Cre; Rosa-RFP; HCN4-GFP, mESCIsl1-RFP)25 on 0.1% (w/v) gelatin coated T25 flasks in 2i medium (870 mL of glascow minimum essential medium (GMEM), 100 mL of fetal bovine serum (FBS), 10 mL of GlutaMAX, 10 mL of non-essential amino a...

<ol>
	<li>Laflamme, M. A., Murray, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration.">Heart regeneration.</a> Nature. 473 (7347), 326-335 (2011).</li><li>Spater, D., Hansson, E. M., Zangi, L., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+cardiomyocyte.">How to make a cardiomyocyte.</a> Development. 141 (23), 4418-4431 (2014).</li><li>Birket, M. J., 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=Pluripotent+stem+cell+derived+cardiovascular+progenitors--a+developmental+perspective.">Pluripotent stem cell derived cardiovascular progenitors--a developmental perspective.</a> Developmental Biology. 400 (2), 169-179 (2015).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., 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=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nature Reviews Molecular Cell Biology. 13 (11), 713-726 (2012).</li><li>Lee, J. H., Protze, S. I., Laksman, Z., Backx, P. H., Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pluripotent+Stem+Cell-Derived+Atrial+and+Ventricular+Cardiomyocytes+Develop+from+Distinct+Mesoderm+Populations.">Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.</a> Cell Stem Cell. 21 (2), 179-194 (2017).</li><li>Protze, S. 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=Sinoatrial+node+cardiomyocytes+derived+from+human+pluripotent+cells+function+as+a+biological+pacemaker.">Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker.</a> Nature Biotechnology. 35 (1), 56-68 (2017).</li><li>Galdos, F. 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=Cardiac+Regeneration:+Lessons+From+Development.">Cardiac Regeneration: Lessons From Development.</a> Circulation Research. 120 (6), 941-959 (2017).</li><li>Lescroart, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+lineage+restriction+in+temporally+distinct+populations+of+Mesp1+progenitors+during+mammalian+heart+development.">Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.</a> Nature Cell Biology. 16 (9), 829-840 (2014).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+networks+in+heart+development+and+regeneration.">Signaling and transcriptional networks in heart development and regeneration.</a> Cold Spring Harbor Perspectives in Biology. 5 (3), 008292 (2013).</li><li>Kelly, R. G., Buckingham, M. E., Moorman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+fields+and+cardiac+morphogenesis.">Heart fields and cardiac morphogenesis.</a> Cold Spring Harbor Perspectives in Medicine. 4 (10), (2014).</li><li>Bruneau, B. 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=Chamber-specific+cardiac+expression+of+Tbx5+and+heart+defects+in+Holt-Oram+syndrome.">Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome.</a> Developmental Biology. 211 (1), 100-108 (1999).</li><li>Watanabe, 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=Fibroblast+growth+factor+10+gene+regulation+in+the+second+heart+field+by+Tbx1,+Nkx2-5,+and+Islet1+reveals+a+genetic+switch+for+down-regulation+in+the+myocardium.">Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (45), 18273-18280 (2012).</li><li>Huynh, T., Chen, L., Terrell, P., Baldini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+Tbx1+expressing+cells+reveals+heterogeneity+in+the+second+cardiac+field.">A fate map of Tbx1 expressing cells reveals heterogeneity in the second cardiac field.</a> Genesis. 45 (7), 470-475 (2007).</li><li>Zhou, 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=Temporally+Distinct+Six2-Positive+Second+Heart+Field+Progenitors+Regulate+Mammalian+Heart+Development+and+Disease.">Temporally Distinct Six2-Positive Second Heart Field Progenitors Regulate Mammalian Heart Development and Disease.</a> Cell Reports. 18 (4), 1019-1032 (2017).</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>Cho, G. S., Tampakakis, E., Andersen, P., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+neonatal+rat+system+as+a+bioincubator+to+generate+adult-like+mature+cardiomyocytes+from+human+and+mouse+pluripotent+stem+cells.">Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells.</a> Nature Protocols. 12 (10), 2097-2109 (2017).</li><li>Bruneau, B. G., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+heart+disease:+entering+a+new+era+of+human+genetics.">Congenital heart disease: entering a new era of human genetics.</a> Circulation Research. 114 (4), 598-599 (2014).</li><li>Liu, 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=The+complex+genetics+of+hypoplastic+left+heart+syndrome.">The complex genetics of hypoplastic left heart syndrome.</a> Nature Genetics. 49 (7), 1152-1159 (2017).</li><li>Li, 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=HAND1+loss-of-function+mutation+contributes+to+congenital+double+outlet+right+ventricle.">HAND1 loss-of-function mutation contributes to congenital double outlet right ventricle.</a> International Journal of Molecular Medicine. 39 (3), 711-718 (2017).</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 (6), 689-698 (2013).</li><li>Uosaki, 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=Direct+contact+with+endoderm-like+cells+efficiently+induces+cardiac+progenitors+from+mouse+and+human+pluripotent+stem+cells.">Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.</a> PLoS One. 7 (10), 46413 (2012).</li><li>Cheng, 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=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140 (12), 2587-2596 (2013).</li><li>Shenje, L. 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=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, 02164 (2014).</li><li>Morita, 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=Sall1+transiently+marks+undifferentiated+heart+precursors+and+regulates+their+fate.">Sall1 transiently marks undifferentiated heart precursors and regulates their fate.</a> Journal of Molecular and Cellular Cardiology. 92, 158-162 (2016).</li><li>Andersen, 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=Precardiac+organoids+form+two+heart+fields+via+Bmp/Wnt+signaling.">Precardiac organoids form two heart fields via Bmp/Wnt signaling.</a> Nature Communications. 9 (1), 3140 (2018).</li><li>Domian, I. 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=Generation+of+functional+ventricular+heart+muscle+from+mouse+ventricular+progenitor+cells.">Generation of functional ventricular heart muscle from mouse ventricular progenitor cells.</a> Science. 326 (5951), 426-429 (2009).</li></ol>

In our protocol, we describe a methodology to generate cardiac spheroids and isolated heart field-specific CPCs. Those can be used to study mechanisms of CPC specification and their properties, as well as for cardiac chamber-specific disease modelling. One previously published work used a mESC line with two fluorescent reporters (Mef2c/Nkx2.5) to study cardiogenesis in vitro, however, both those markers are expressed at embryonic day 9.5-10 when cardiomyocytes are already formed26. To our knowledg...

The use of pluripotent stem cells (PSCs) has revolutionized the field of cardiac regeneration and personalized medicine with patient-specific myocytes for disease modeling and drug therapies1,2,3,4. More recently, in vitro protocols for the generation of atrial vs ventricular as well as pacemaker-like PSC-derived cardiomyocytes have been developed5,6. However, whether cardiogenesis can be recreated in vitro to study cardiac development and subsequently generate ventricular chamber-specific cardiac cells is still unclear.
During early embryonic development, mesodermal cells under the influence of secreted morphogens such as BMP4, Wnts and Activin A form the primitive streak7. Cardiac mesodermal cells marked by the expression of Mesp1, migrate anteriorly and latterly to form the cardiac crescent and then the primitive heart tube7,8. This migratory group of cells includes two very distinct populations of cardiac progenitor cells (CPCs), namely the first and the second heart field (FHF and SHF)9,10. Cells from the SHF are highly proliferative and migratory and are primarily responsible for the elongation and looping of the heart tube. Additionally, SHF cells differentiate to cardiomyocytes, fibroblasts, smooth muscle and endothelial cells as they enter the heart tube to form the right ventricle, right ventricular outflow tract and large part of both atria7,10. In contrast, FHF cells are less proliferative and migratory and differentiate mainly to cardiomyocytes as they give rise to the left ventricle and a smaller part of the atria11. Moreover, SHF progenitors are marked by the expression of Tbx1, FGF8, FGF10 and Six2 while FHF cells express Hcn4 and Tbx511,12,13,14,15.
PSCs can differentiate to all three germ layers and subsequently to any cell type in the body4,16. Therefore, they offer tremendous potential for understanding heart development and for modelling specific developmental defects resulting in congenital heart disease, the most frequent cause of birth defects17. A large subgroup of congenital heart disease includes chamber-specific cardiac abnormalities18,19. However, it still unclear whether these originate from anomalous heart field development. In addition, given the inability of cardiomyocytes to proliferate after birth, there have been extensive efforts to create cardiac tissue for heart regeneration1,7,20. Considering the physiological and morphological differences between cardiac chambers, generation of chamber-specific cardiac tissue using PSCs is of significant importance. While recent advances in developmental cardiology have led to robust generation of cardiac cells from PSCs, it is still unclear if the two heart fields can be induced in PSC systems.
To recapitulate cardiogenesis in vitro and study the specification and properties of CPCs, we previously used a system based on differentiating PSC-derived cardiac spheroids21,22,23,24. Recently, we generated mouse embryonic stem cells (mESCs) with GFP and RFP reporters under the control of the FHF gene Hcn4 and the SHF gene Tbx1, respectively (mESCsTbx1-Cre; Rosa-RFP; HCN4-GFP) 25. In vitro differentiated mESCs formed cardiac spheroids in which GFP+ and RFP+ cells appeared from two distinct areas of mesodermal cells and patterned in a complementary manner. The resulting GFP+ and RFP+ cells exhibited FHF and SHF characteristics, respectively, determined by RNA-sequencing and clonal analyses. Importantly, using mESCs carrying the Isl1-RFP reporter (mESCIsl1-RFP), we discovered that SHF cells were faithfully marked by the cell-surface protein CXCR4, and this can enable isolation of heart field-specific cells without transgenes. The present protocol will describe the generation and isolation of heart field-specific CPCs from mESCs, which may serve as a valuable tool for studying chamber-specific heart disease.

<table border="0" cellpadding="0" cellspacing="0" style="width:1251px;" width="1250">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">&#946;-mercaptoethanol</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:344px;">M6250</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.1% (w/v) Gelatin</td>
			<td>EMD Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td align="right">11360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100X Pen/Strep</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1X PBS w/o Calcium and Magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20% Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-980-493</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">5 ml Polystyrene round-bottom tube with a 40&#956;m cell strainer</td>
			<td>BD Falcon</td>
			<td align="right">35223</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Activin A</td>
			<td>R &#38; D Systems</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A-4544</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27 minus vitamin A (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">12587010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BMP4</td>
			<td>R &#38; D Systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">Cell sorter</td>
			<td style="width:245px;">Sony</td>
			<td style="width:344px;">SH800</td>
			<td style="width:241px;">Sony or any other fluorescence-activated cell sorter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">Cell strainer 70&#956;m</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge Sorvall Legend XT</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">75004508</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CHIR99021</td>
			<td>Selleck chemicals</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">CO2 Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">51030285</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Corning Ultra Low Attachment T75 flask</td>
			<td>Corning</td>
			<td>07-200-875</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Countless II FL automated cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31571, Lot #1757130</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle&#39;s Medium high glucose (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ESGRO (LIF)</td>
			<td>Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOS FL microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF4300</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glasgow&#8217;s MEM (GMEM)</td>
			<td>Gibco</td>
			<td align="right">11710035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (100 x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ham&#8217;s F12</td>
			<td>Gibco</td>
			<td>10-080-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM</td>
			<td>Gibco</td>
			<td align="right">12440053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monothioglycero (MTG)</td>
			<td>Sigma</td>
			<td>M-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Troponin T antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MS-295-P1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2-SUPPLEMENT</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential amino acid solution (NEAA</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD0325901</td>
			<td>Selleckchem</td>
			<td>S1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-efluor 710 conjugated anti-Cxcr4 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-9991-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suspension culture dish 150 mm x 25mm</td>
			<td>Corning</td>
			<td align="right">430597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 flasks</td>
			<td>Corning</td>
			<td align="right">353109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE (Trypsin)</td>
			<td>Gibco</td>
			<td align="right">12604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 (ROCK inhibitor)</td>
			<td>Stem cell technologies</td>
			<td align="right">72304</td>
			<td></td>
		</tr>
	</tbody>
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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.

After approximately 132 h of differentiation, Tbx1-RFP and Hcn4-GFP CPCs can be detected using a fluorescent microscope (Figure 2). Generally, GFP and RFP cells appear approximately around the same time. The two populations of CPCs continue to expand in close proximity and commonly in a complementary pattern. Adjusting the concentrations of Activin A and BMP4 will alter the percentages of FHF vs SHF CPCs (Figure 3). CPC specification in vitro was primarily deter...

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In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

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 ...

Research

JoVE Journal

Developmental Biology

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 ...

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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 ...

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...