Name: differentiation of atrial cardiomyocytes from pluripotent stem cells using the bmp antagonist grem2
Uploaded: 2016-03-10T15:00:00
Description: Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

This work was supported by NIH grants HL083958 and HL100398 (A.K.H.) and 2T32HL007411-33 &#34;Program in Cardiovascular Mechanisms: Training in Investigation&#34; (J.B.).

1. Preparation of Cell Culture Media, Solutions, and Reagents.
<ol>
	<li>Prepare 500 ml of Mouse embryonic stem cell (mESC) media by mixing and sterile filtering (0.2 &#956;m pore size) 445 ml Glasgow Minimum Essential Medium (GMEM), 50 ml heat inactivated fetal bovine serum (FBS), 5 ml 100X concentrated L-Glutamine replacement, 5 &#181;l recombinant mouse Leukemia Inhibitory Factor (LIF) at 1 x 107 units per ml, and 1.43 &#181;l &#223;-Mercaptoethanol (final concentration is 50 &#181;M). Store at 4 &#730;C ...

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	<li>Loya, K., Eggenschwiler, 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=Hepatic+differentiation+of+pluripotent+stem+cells.">Hepatic differentiation of pluripotent stem cells.</a> Biological Chemistry. 390 (10), 1047-1055 (2009).</li><li>Narazaki, G., 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=Directed+and+systematic+differentiation+of+cardiovascular+cells+from+mouse+induced+pluripotent+stem+cells.">Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells.</a> Circulation. 118 (5), 498-506 (2008).</li><li>Zhang, Y., Pak, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+single-step+induction+of+functional+neurons+from+human+pluripotent+stem+cells.">Rapid single-step induction of functional neurons from human pluripotent stem cells.</a> Neuron. 78 (5), 785-798 (2013).</li><li>Park, I. H., Arora, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134 (5), 877-886 (2008).</li><li>Golos, T. G., Giakoumopoulos, M., Garthwaite, M. A. <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+cells+as+models+of+trophoblast+differentiation:+progress,+opportunities,+and+limitations.">Embryonic stem cells as models of trophoblast differentiation: progress, opportunities, and limitations.</a> Reproduction. 140 (1), 3-9 (2010).</li><li>Boheler, K. R., Czyz, J., Tweedie, D., Yang, H. T., Anisimov, S. V., Wobus, A. M. <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>Burridge, P. W., Anderson, 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=Improved+human+embryonic+stem+cell+embryoid+body+homogeneity+and+cardiomyocyte+differentiation+from+a+novel+V-96+plate+aggregation+system+highlights+interline+variability.">Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability.</a> Stem Cells. 25 (4), 929-938 (2007).</li><li>Cai, C. L., Liang, 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=Isl1+Identifies+a+cardiac+progenitor+population+that+proliferates+prior+to+differentiation+and+contributes+a+majority+of+cells+to+the+heart.">Isl1 Identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart.</a> Developmental Cell. 5 (6), 877-889 (2003).</li><li>Fujiwara, M., Yan, 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=Induction+and+enhancement+of+cardiac+cell+differentiation+from+mouse+and+human+induced+pluripotent+stem+cells+with+Cyclosporin-A.">Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with Cyclosporin-A.</a> PLoS ONE. 6 (2), e16734 (2011).</li><li>Rai, M., Walthall, J. M., Hu, J., Hatzopoulos, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+antagonism+by+Dkk1+counter+activates+canonical+wnt+signaling+and+promotes+cardiomyocyte+differentiation+of+embryonic+stem+cells.">Continuous antagonism by Dkk1 counter activates canonical wnt signaling and promotes cardiomyocyte differentiation of embryonic stem cells.</a> Stem Cells and Development. (1), 54-66 (2012).</li><li>Burridge, P. W., Matsa, 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=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature Methods. 11 (8), 855-860 (2014).</li><li>Lian, X., Zhang, 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/&#946;-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/&#946;-catenin signaling under fully defined conditions.</a> Nature Protocols. 8 (1), 162-175 (2013).</li><li>Zhang, J., Klos, 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=Extracellular+matrix+promotes+highly+efficient+cardiac+differentiation+of+human+pluripotent+stem+cells:+the+matrix+sandwich+method.">Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.</a> Circulation Research. 111 (9), 1125-1136 (2012).</li><li>Tanwar, V., Bylund, J. 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=Gremlin+2+promotes+differentiation+of+embryonic+stem+cells+to+atrial+fate+by+activation+of+the+JNK+signaling+pathway.">Gremlin 2 promotes differentiation of embryonic stem cells to atrial fate by activation of the JNK signaling pathway.</a> Stem Cells. 32 (7), 1774-1788 (2014).</li><li>Kattman, S. J., Witty, A. 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=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>M&#252;ller, I. I., Melville, D. 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=Functional+modeling+in+zebrafish+demonstrates+that+the+atrial-fibrillation-associated+gene+GREM2+regulates+cardiac+laterality,+cardiomyocyte+differentiation+and+atrial+rhythm.">Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm.</a> Disease Models &amp; Mechanisms. 6 (2), 332-341 (2013).</li><li>Kattamuri, C., Luedeke, D. M., Thompson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+recombinant+protein+related+to+DAN+and+cerberus+(PRDC).">Expression and purification of recombinant protein related to DAN and cerberus (PRDC).</a> Protein Expression and Purification. 82 (2), 389-395 (2012).</li><li>Schulz, H., Kolde, 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=The+FunGenES+Database:+A+genomics+resource+for+mouse+embryonic+stem+cell+differentiation.">The FunGenES Database: A genomics resource for mouse embryonic stem cell differentiation.</a> PLoS ONE. 4 (9), e6804 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-buffered+saline+(PBS).">Phosphate-buffered saline (PBS).</a> Cold Spring Harbor Protocols. , (2006).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+Real-Time+quantitative+PCR+and+the+2&#8722;&#916;&#916;CT+method.">Analysis of relative gene expression data using Real-Time quantitative PCR and the 2&#8722;&#916;&#916;CT method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Beck, H., Semisch, M., Culmsee, C., Plesnila, N., Hatzopoulos, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egr-1+regulates+expression+of+the+glial+scar+component+phosphacan+in+astrocytes+after+experimental+stroke.">Egr-1 regulates expression of the glial scar component phosphacan in astrocytes after experimental stroke.</a> American Journal of Pathology. 173 (1), 77-92 (2008).</li><li>Graham, E. L., Balla, C., Franchino, H., Melman, Y., del Monte, F., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation, culture, and functional characterization of adult mouse cardiomyoctyes.</a> Journal of Visualized Experiments. (79), (2013).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recording+of+ion+channels+expressed+in+Xenopus+oocytes.">Patch clamp recording of ion channels expressed in Xenopus oocytes.</a> Journal of Visualized Experiments. (20), (2008).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> Journal of Visualized Experiments. (20), (2008).</li><li>Sachinidis, A., Fleischmann, B. K., Kolossov, E., Wartenberg, M., Sauer, H., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+specific+differentiation+of+mouse+embryonic+stem+cells.">Cardiac specific differentiation of mouse embryonic stem cells.</a> Cardiovascular Research. 58 (2), 278-291 (2003).</li><li>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=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Current Opinion in Cell Biology. 7 (6), 862-869 (1995).</li><li>H&#246;pfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.+Volume+2:+molecular+embryo+analysis,+live+imaging,+transgenesis,+and+cloning.">Differentiating embryonic stem cells into embryoid bodies. Volume 2: molecular embryo analysis, live imaging, transgenesis, and cloning.</a> Methods in Molecular Biology. , 254-279 (2004).</li><li>Ao, A., Williams, C. H., Hao, J., Hong, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+mouse+embryonic+stem+cell+based+assay+for+quantifying+cardiogenic+Induction+Efficiency.">Modified mouse embryonic stem cell based assay for quantifying cardiogenic Induction Efficiency.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Antonchuk, J., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+embryoid+bodies+from+human+pluripotent+stem+cells+using+AggreWell&#8482;+plates.">Formation of embryoid bodies from human pluripotent stem cells using AggreWell&#8482; plates.</a> Basic Cell Culture Protocols. 946, 523-533 (2013).</li><li>Horii, T., Nagao, Y., Tokunaga, T., Imai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells.">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li></ol>

This protocol routinely produces cultures with a high percentage of CMs that are characteristic of the atrial lineage. As with any differentiation protocol, the quality of the mESCs prior to differentiation should be given particular attention. mESCs should be routinely monitored for proper morphology (Figure 1A). Any spontaneous differentiation that occurs prior to formation of EBs will severely limit the efficiency of cardiogenesis and should be removed before passaging (Figure 1B). EB...

Pluripotent stem cells are a powerful tool for generating and studying cells from a host of difficult to access tissues for basic research and pre-clinical studies, especially in humans1-5. Proper modulation of developmental signaling pathways can direct the differentiation of pluripotent stem cells to the desired phenotypic fate. Many protocols have been developed to generate cardiomyocytes (CMs) from pluripotent stem cells6-14. These protocols generally involve modulation of TFG&#946; superfamily (Activin, BMP, and TGF&#946;) and Wnt pathways through timed addition of exogenous growth factors and/or small molecules10,12-15. These protocols are generally effective at increasing the percentage of cells that become CMs, but lack specificity, generating a mixed population of cells representing atrial, ventricular, and nodal/conduction system lineages. In order to study specific cardiac subtypes a more directed differentiation approach is required.
Gremlin2 (Grem2, also called Protein Related to Dan and Cerberus or PRDC for short) is a secreted BMP antagonist that is necessary&#160;for proper cardiac differentiation and atrial chamber formation during cardiac development in zebrafish16. Treating differentiating embryonic stem cells with Grem2 at differentiation day 4, just after peak expression of mesodermal markers T-Brachyury and Cerberus like 1, increases the yield of CMs and generates a pool of cells predominantly of the atrial lineage14.
Recombinant Grem2 is used to treat the differentiating cells and can be made using standard protein production techniques17 or can be purchased commercially. It is highly soluble in aqueous solutions and may be added exogenously to cultures at the desired time point.
Differentiation can be tracked using RT-qPCR to quantify expression of markers representative of cardiovascular progenitors, cardiac progenitors, and committed CMs. Immunofluorescence can also be used to identify and visualize spatial distribution of cardiac cell types.
Applications that require pure populations are more readily carried out when using a reporter system to identify and isolate CMs. For this purpose, we have introduced the &#945;MHC-DsRedNuc construct into the mouse CGR8 ES cell line14. CGR8 cells grow and remain pluripotent without feeder cells, facilitating expansion and differentiation assays18. The ES cell line contains a DSRed fluorescent protein coding sequence with a nuclear localization signal under the cardiac-specific alpha Myosin Heavy Chain (&#945;Mhc or Myh6) gene promoter. Using these cells, CMs can be easily identified and isolated for quantification,&#160;cell sorting,&#160;electrophysiology, drug screens, and studying mechanisms of atrial differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="479">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:217px;">GMEM</td>
			<td style="width:116px;">Life Technologies</td>
			<td style="width:83px;">11710</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Life Technologies</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamax</td>
			<td>Life Technologies</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIF</td>
			<td>EMD Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#223;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Sigma</td>
			<td>I3390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-Essential Amino Acids</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Tissue Culture Plates</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Bacterial Petri Dishes</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 cm Bacterial Petri Dishes</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well tissue culture plates</td>
			<td>Sarstedt</td>
			<td>83.3920</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gremlin 2 recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>2069-PR-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile filter units</td>
			<td>Thermo Fisher</td>
			<td>09-741-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin (from porcine skin)</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X PBS, Sterile</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA&#160;</td>
			<td>Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% Trypsin-EDTA solution</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no Calcium, no Magnesium</td>
			<td>Life Technologies</td>
			<td>14200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Prior to differentiation, pluripotent stem cells should be compact and free of spontaneous differentiation. The cells shown in Figure 1A are ready to be singularized and used for hanging drops as described in 5.1 of the protocol section. The cells shown in Figure 1B are spontaneously differentiating and should not be used for preparation of hanging drops.
The panels included in Figure 2<...

Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

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

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 ...

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