The authors acknowledge funding agencies, the IMI StemBANCC European community programme, the leducq Foundation (SHAPEHEART) and the Agence Nationale de la Recherche (Genopath)

1. DNA-protein Cross-linking
<ol>
	<li>Fix in 15 ml tubes harvested-ES cells (2 x&#160;106 cells for regular ChIP, 2 x&#160;105 cells for microChIP), embryoid bodies (EBs) generated from ES cells and embryonic heart tissues dissected from E9.5 mouse embryos (atrioventricular canal, outflow tract and ventricle) using 1% formaldehyde in PBS for cells or in permeabilization PB2 buffer for embryonic tissues. Place the tubes on orbital shaker at a speed of 60 rpm at room temperature for exactly 10 min.</li>
	<li>Stop the cross-linking reaction by adding glycine to a final concentration of 125 mM and incubate for 5 min at room temperature on the orbital shaker at a speed of 60 rpm.</li>
</ol>
2. Cell Lysis and Chromatin Fragmentation
<ol>
	<li>Wash twice cross-linked cells resuspended in 10 ml PBS 1x or embryonic tissues resuspended in 1 ml PB2. Centrifuge at 1,000 x g for 5 min at 4 &#176;C. Discard the supernatants.</li>
	<li>Add protease inhibitors to buffer PB1 or PB2 (2 &#181;g/ml Leupeptin, 1 &#181;g/ml Aprotinin, and 0.1 mM PMSF) (Table 1). Resuspend the cell pellet or embryonic tissue from step 2.1 in 1 ml of Buffer PB1 or PB2 respectively. Pipet up and down and resuspend the cells or tissues. Use a 1 ml syringe with a 21 gauge needle to homogenize cells or tissue. Incubate at 4 &#176;C (on a wheel) for 10 min.</li>
	<li>Spin down at 3,000 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in 300 &#181;l of respective SB buffers (Table 1) supplemented with protease inhibitors in a clean tube (certified RNase-, DNase-, and pyrogen-free) to prevent any DNA degradation. Incubate for 15 to 30 min on ice.</li>
	<li>Sonicate the samples at 4 &#176;C to shear the chromatin using a sonicator according to programs listed in Table 2 for each material. Adjust sonication duration depending upon the next application (i.e., PCR or sequencing). Sheared DNA size should be around 500 bp for PCR and 300 bp for sequencing.</li>
	<li>Centrifuge sonicated cell lysate for 10 min at 6,000 x g at 4 &#176;C, transfer the supernatant (chromatin) in a clean 1.5 ml tube and discard the pellet.</li>
	<li>Dilute the chromatin in solution (supernatant buffer B) for 10 times with water and assess the optical density using 1.5 &#181;l in a nano-spectrophotometer. Obtain the concentration of proteins in &#181;g/&#181;l using the following formula: 1.55 x OD 280 - 0.76 x OD260.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Utilization</td>
			<td colspan="4">Composition</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td rowspan="2">A</td>
			<td rowspan="2">Permeabilization</td>
			<td colspan="4">PB1&#160;: 5 mM PIPES pH 8; 85 mM KCl; 0.5 % NP40</td>
			<td>All</td>
		</tr>
		<tr>
			<td colspan="4">PB2&#160;: 15 mM HEPES pH 7.6, 15 mM NaCl, 4 mM MgCl2 60 mM KCl, 0.5% TRITON X-100</td>
			<td>Embryonic tissue</td>
		</tr>
		<tr>
			<td rowspan="3">B</td>
			<td rowspan="3">Lysis /Sonication</td>
			<td colspan="4">SB1: 1 % SDS; 10 mM EDTA; 50 mM Tris-HCl pH 8</td>
			<td>ESC</td>
		</tr>
		<tr>
			<td colspan="4">SB2: 50 mM HEPES-KOH pH7.9&#160;; 140 mM; 1mM&#160; EDTA; 0.1% deoxycholate; 0.1 % SDS</td>
			<td>EBs</td>
		</tr>
		<tr>
			<td colspan="4">SB3:15 mM HEPES pH7.6, 15 mM NaCl&#160;; 60 mM KCl, 1mM EDTA, 0.5 mM EGTA&#160;; 1% TRITON-X100, 0&#160;.1% SDS , 0.5% laurylsarcosine</td>
			<td>Embryonic tissue</td>
		</tr>
		<tr>
			<td>C</td>
			<td>ChIP buffer</td>
			<td colspan="4">150 mM NaCl&#160;; 50 mM Tris-HCl pH 7.5&#160;; 5 mM EDTA&#160;; 0.5 % NP40&#160;; 1% TRITON-X100 .</td>
			<td>All</td>
		</tr>
		<tr>
			<td>D</td>
			<td>DNA/protein Elution</td>
			<td colspan="4">D1&#160;: 1 % SDS; 100 mM NaHCO3 
			D2: 50 mM Tris pH 7.6 /5 mM EDTA, 15 mM DTT, 2% SDS</td>
			<td></td>
		</tr>
		<tr>
			<td>E</td>
			<td>DNA binding beads preparation</td>
			<td colspan="4">E&#160;: 20 % PEG 8000; 2.5 M NaCl; 10 mM Tris-HCl pH 8; 1mM EDTA</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. ChIP buffers.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Sonication program</td>
		</tr>
		<tr>
			<td>Embryonic stem cells</td>
			<td>15 cycles of 30 sec ON and 30 sec OFF</td>
		</tr>
		<tr>
			<td>Embryoid bodies</td>
			<td>30 cycles of 30 sec ON and 30 sec OFF</td>
		</tr>
		<tr>
			<td>Embryonic tissues</td>
			<td>21 cycles of 30 sec ON and 30 sec OFF</td>
		</tr>
	</tbody>
</table>
Table 2. Sonication Programs.
3. Immunoprecipitation and Washes
<ol>
	<li>Wash 3 times Protein A-conjugated beads with buffer C . Take 100 &#181;l of beads in a clean 1.5 ml tube and add 1 ml of buffer C (Table 1). The tube is then transferred onto a magnet; wait for 1 min and aspirate the supernatant. Repeat twice the washing step.</li>
	<li>Incubate antibody raised against modified histones or transcription factor (concentrations listed in Table 3) with 20 &#181;l of washed Protein A conjugated beads and 1 ml of buffer C supplemented with protease inhibitors in a clean 1.5 ml tube. Set the samples on a rotator at 40 rpm for at least 2 hr at 4 &#176;C.</li>
	<li>Wash antibody-beads complexes 3 times with 1 ml of buffer C (manipulation described in 3.1).</li>
	<li>Prepare 2 tubes, one containing 150 &#181;g of chromatin and antibody-beads complexes and the other tube containing 150 &#181;g of chromatin and 20 &#181;l of washed beads; Add 1 ml of buffer C supplemented with protease inhibitors to each tube and incubate the samples on a rotating wheel at 40 rpm overnight at 4 &#176;C. 
	NOTE: The concentration of chromatin must be at least 300 &#181;g to immunoprecipitate a transcription factor or for sequential ChIP.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standard</td>
			<td>&#160;concentration (ng/&#181;l)</td>
			<td>Volume (&#181;l)</td>
			<td>Total DNA concentration (ng)</td>
		</tr>
		<tr>
			<td>A</td>
			<td>10.0</td>
			<td>1</td>
			<td>10.0</td>
		</tr>
		<tr>
			<td>B</td>
			<td>5.00</td>
			<td>1</td>
			<td>5.00</td>
		</tr>
		<tr>
			<td>C</td>
			<td>2.50</td>
			<td>1</td>
			<td>2.50</td>
		</tr>
		<tr>
			<td>D</td>
			<td>1.25</td>
			<td>1</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>E</td>
			<td>0.625</td>
			<td>1</td>
			<td>0.625</td>
		</tr>
		<tr>
			<td>F</td>
			<td>0.3125</td>
			<td>1</td>
			<td>0.3125</td>
		</tr>
		<tr>
			<td>G</td>
			<td>0.156</td>
			<td>1</td>
			<td>0.156</td>
		</tr>
		<tr>
			<td>H</td>
			<td>0.0</td>
			<td>1</td>
			<td>0.0</td>
		</tr>
	</tbody>
</table>
Table 3. Standards concentrations.
4. DNA Elution, Cross-link Reversal and Proteinase K Digestion
<ol>
	<li>Set the samples in the magnetic rack. Recover the supernatant containing unbound antibody chromatin. 
	NOTE: Samples can be quickly frozen in liquid nitrogen at that step and stored at -80 &#176;C. Chromatin can be used in other immunoprecipitation assays with other antibody. Wash each sample 3 times with 1 ml of buffer C (manipulation described before).</li>
	<li>Elute the antibody-bound protein by adding 150 &#181;l of buffer D1 or D2 if sequential ChIP has to be done (Table 1) to washed ChIP materials and incubate the samples for 20 min at 50 &#176;C in a heating block.</li>
	<li>Remove tubes from the heating block and put samples in the magnetic rack, recover the supernatant in a clean 1.5 ml tube (certified RNase-, DNase-, and pyrogen-free) and discard the beads.</li>
	<li>Use supernatant for a sequential ChIP adding 1 to 3 &#181;g of the second antibody in 2 volumes of buffer C or reverse crosslink by adding 5 M NaCl to obtain a final concentration of 200 mM.</li>
	<li>Prepare an input sample of chromatin in a clean 1.5 ml tube (certified RNase-, DNase-, and pyrogen-free) taking the same amount of chromatin as that of the IP sample (150 &#181;g) in a final volume of 150 &#181;l of water (RNase DNase free water). Add 5M NaCl to a final concentration of 200 mM. Incubate samples overnight at 65 &#176;C.</li>
	<li>Next day, remove samples from the heating block, add 250 mM EDTA to obtain a final concentration of 12.5 mM and proteinase K to obtain a final concentration of 250 &#181;g/ml in the ChIP material and digest at 55 &#176;C for 2 hr in the heating block.</li>
</ol>
5. DNA Isolation Using DNA-binding Beads
<ol>
	<li>Preparation of beads
	<ol>
		<li>Start to bring the beads at room temperature for at least 30 min, vortex the beads very thoroughly. Beads settle down, so work fast when pipetting. Transfer 1 ml of beads in clean 1.5 ml tube (certified RNase-, DNase-, and pyrogen-free).</li>
		<li>Set on magnet, wait for 2 - 3 min for the solution to clarify, discard supernatant. Remove from magnet, add 1 ml of 0.5 M EDTA and mix by brief vortexing.</li>
		<li>Repeat step 5.1.2 twice, and discard supernatant at the end.</li>
		<li>Remove from magnet, add 1 ml of buffer E and slowly resuspend the beads. Transfer the whole mix (beads in buffer E) into 50 ml of buffer E. Place on a shaker and let it mix slowly at room temperature for 1 hr.</li>
		<li>Test DNA-binding beads following the purification protocol described below using 3 experimental conditions: 50 &#181;l of beads/ 50 &#181;l of DNA (1 volume/1 volume), 100 &#181;l of beads/50 &#181;l of DNA (2 volumes/1 volume) and 125 &#181;l of beads/ 50 &#181;l of DNA (2.5 volumes/1 volume). Let migrate purified DNA on a 1.5% agarose gel to check the size of the fragments (Figure 1A).</li>
	</ol>
	</li>
	<li>DNA Purification
	<ol>
		<li>Add 425 &#181;l (2.5 volumes) of DNA binding magnetic beads to each DNA sample (about 170 &#181;l). Resuspend slowly by pipetting up and down (about 10 times) and incubate at room temperature for 10 min.</li>
		<li>Place on magnet for 5 min, then aspirate supernatant and discard (on magnet).</li>
		<li>Add 600 &#181;l of freshly made 80% ethanol to the tube on magnet, wait for 1 min, aspirate supernatant and discard and then repeat the previous step once more.</li>
		<li>Give a quick spin, for 5 sec (microfuge), place the tube on magnet and remove the last drops of ethanol. Let dry for 1 min at RT</li>
		<li>Elute the DNA by adding 20 &#181;l of DNase RNase free water. Remove from magnet and vortex the sample.</li>
		<li>Incubate for 3 min at room temperature and then place samples on magnet. Aspirate eluted DNA into a new tube (certified RNase-, DNase-, and pyrogen-free).</li>
		<li>Run the DNA from the input fraction on a 1.5% agarose gel to check the size of DNA fragments (Figure 1B).</li>
	</ol>
	</li>
</ol>
6. DNA Quantification Using a Fluorescence Detection Instrument
<ol>
	<li>Serially dilute in water 1 &#956;g DNA (1 kb ladder) to give a total of 7 dilutions plus a no-DNA point (dilutions gathered in Table 3).</li>
	<li>Prepare a solution of PCR Green I dye, sufficient for all samples (purified DNA and standard dilutions A-H): dilute 10,000x the Green dye in 1x TE buffer.</li>
	<li>Add 1 &#181;l of each DNA samples (purified DNA and standard dilutions A-H) to 10 &#181;l 1x green dye mix in capillaries cuvettes to be read in a fluorimeter at 488 nm wavelength. For the no-DNA assay, use 1 &#181;l of the 1x TE buffer.</li>
	<li>Build a calibration curve using a graphic software and determine the relative concentrations to the range of DNA samples in ng/&#181;l. The DNA is then ready for PCR or to make sequencing libraries.</li>
</ol>
7. PCR
<ol>
	<li>Selection of primers
	<ol>
		<li>Design primers of interest to interrogate the genomic regions of interest. Design primers to amplify enhancer regions of genes using the UCSC genome browser. Enhancers are further predicted using H3K4me1 or p300 ChIP-seq9 data available in GEO data set.</li>
	</ol>
	</li>
	<li>PCR
	<ol>
		<li>Run PCR reactions for 45 cycles (8 sec 95 &#176;C, 8 sec 60 &#176;C, 8 sec 72 &#176;C) using a real-time PCR thermocycler in 25 &#956;l green dye mix, 2 ng DNA, and 0.5 &#956;l primer mix (20 &#956;M stock solution)10.</li>
	</ol>
	</li>
	<li>Analysis of enrichment
	<ol>
		<li>Calculate Absolute enrichment assuming that at most 1% of nucleosome were immunoprecipitated11.</li>
		<li>Consider the genomic as enriched if 10 ng IP samples show a greater enrichment when compared to 0.1 ng of input DNA.</li>
		<li>Express results as the fold increase in enrichment over a non-enriched region after normalization to the input and adjustment with a non-specific control sample.</li>
		<li>Consider the input DNA to be diluted by 100 (dilution factor or DF).</li>
		<li>Normalize the input using the following equation 2-&#916;Ct&#160;x 100%, where -&#916;Ct = Ct[IP] - Ct[Input x DF].</li>
		<li>Adjust control using the following equation (&#916;Ct[IP]-&#916;Ct[NS]) where NS is the non-specific sample (no antibody IP, or IgG IP).</li>
		<li>Calculate the fold enrichment of the sample as 2-&#916;&#916;Ct. A file including all steps of calculations with formulas in which only each PCR sample can be entered will facilitate the analysis of results.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6, 826-835 (2005).</li><li>Chen, T., Dent, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifiers+and+remodellers:+regulators+of+cellular+differentiation.">Chromatin modifiers and remodellers: regulators of cellular differentiation.</a> Nat Rev Genet. 15, 93-106 (2014).</li><li>Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+chromatin+immunoprecipitation.">The current state of chromatin immunoprecipitation.</a> Mol Biotechnol. 45, 87-100 (2010).</li><li>Gade, P., Kalvakolanu, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay+as+a+tool+for+analyzing+transcription+factor+activity.">Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.</a> Methods Mol Biol. 809, 85-104 (2012).</li><li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+retardation+assay.">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li><li>Van Vliet, P., Wu, S. M., Zaffran, S., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+cardiac+development:+a+view+from+stem+cells+to+embryos.">Early cardiac development: a view from stem cells to embryos.</a> Cardiovasc Res. 96, 352-362 (2012).</li><li>Leschik, J., Caron, L., Yang, H., Cowan, C., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+bivalent+epigenetic+marks+in+two+human+embryonic+stem+cell+lines+reveals+a+different+cardiogenic+potential.">A view of bivalent epigenetic marks in two human embryonic stem cell lines reveals a different cardiogenic potential.</a> Stem Cells Dev. 24, 384-392 (2015).</li><li>Bonn, S., Zinzen, R. P., Girardot, C., Gustafson, E. H., Perez-Gonzalez, A., Delhomme, N., Ghavi-Helm, Y., Wilczynski, B., Riddell, A., Furlong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+analysis+of+chromatin+state+identifies+temporal+signatures+of+enhancer+activity+during+embryonic+development.">Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.</a> Nat Genet. 44, 148-156 (2012).</li><li>Kim, T. K., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+Functional+Commonalities+between+Enhancers+and+Promoters.">Architectural and Functional Commonalities between Enhancers and Promoters.</a> Cell. 162, 948-959 (2015).</li><li>Abboud, N., Moore-Morris, T., Hiriart, E., Yang, H., Bezerra, H., Gualazzi, M. G., Stefanovic, S., Guenantin, A. C., Evans, S. M., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cohesin-OCT4+complex+mediates+Sox+enhancers+to+prime+an+early+embryonic+lineage.">A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage.</a> Nat Commun. 6, 6749 (2015).</li><li>Dahl, J. A., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q2ChIP,+a+quick+and+quantitative+chromatin+immunoprecipitation+assay,+unravels+epigenetic+dynamics+of+developmentally+regulated+genes+in+human+carcinoma+cells.">Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells.</a> Stem Cells. 25, 1037-1046 (2007).</li><li>Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bivalent+chromatin+structure+marks+key+developmental+genes+in+embryonic+stem+cells.">A bivalent chromatin structure marks key developmental genes in embryonic stem cells.</a> Cell. 125, 315-326 (2006).</li><li>Wamstad, J. A., Alexander, J. M., Truty, R. M., Shrikumar, A., Li, F., Eilertson, K. E., Ding, H., Wylie, J. N., Pico, A. R., Capra, 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, 206-220 (2012).</li><li>Stergachis, A. B., Neph, S., Reynolds, A., Humbert, R., Miller, B., Paige, S. L., Vernot, B., Cheng, J. B., Thurman, R. E., Sandstrom, 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=Developmental+fate+and+cellular+maturity+encoded+in+human+regulatory+DNA+landscapes.">Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.</a> Cell. 154, 888-903 (2013).</li><li>Brind'Amour, J., Liu, S., Hudson, M., Chen, C., Karimi, M. M., Lorincz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-low-input+native+ChIP-seq+protocol+for+genome-wide+profiling+of+rare+cell+populations.">An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations.</a> Nat Commun. 6, 6033 (2015).</li></ol>

The authors have nothing to disclose.

Epigenetics has become an important field of research in developmental biology. How a genetic program is activated in embryonic cells to allow the cells to acquiring a specific identity within an embryonic lineage has been for long time a key question for developmental biologists.
ChIP has been broadly used within the last years and combined to DNA sequencing following improvement in resolution of sequencing. This has become a powerful technique in order to investigate in a genome wide dependent manner the epigenetic landscape of cells or specific embryonic and adult tissues. The ChIP assay buffers and sonication conditions have been greatly improved to allow assays for small amount of biological material. This includes specific regions of embryonic hearts obtained by microdissection or by FACS-sorted cells expressing a fluorescent reporter protein. Indeed ChIP performed on heterogeneous cell populations 1314 can lead to misleading results that are difficult to interpret. The epigenetic signature marks very accurately cell populations. That signature confers to them a specific potential of differentiation and determines their fate. It is thus important to work with purified (sorted) cell populations or dissected embryonic tissues.
We have described above robust protocols in order to achieve these goals. We have used these protocols for ChIP-PCR, ChIP on ChIP7, 10 or ChIP-sequencing (Figure 4, manuscript in preparation). The reason for robustness lays on the permeabilization and cell lysis buffers that have been optimized to ensure a complete cell and nuclear lysis and efficient chromatin sonication. The protocols can thus be used for small amount of embryonic tissues.
Limitations of these protocols are inherent to ChIP. More specifically, the spatial resolution is not very high with DNA sizes around 500 bp. However, it allows us to investigate the dynamics of bivalent domains in differentiating ES cells7 and to reveal differences in enrichment of modified histones on EMT gene regulatory regions in wild type or gene-mutated cells. (Figure 2). Deep sequencing of immunoprecipitated DNA (Figure 4) partially overcomes this limitation.
Enrichment of a specific genomic region following immunoprecipitation of a transcription factor does not indicate for sure that the factor directly binds DNA. It could only be part of complex factory bound to DNA. This is an important point to be aware about. In contrast, gel retardation assay could be used to address the question.
Having the technology in hands combined with deep sequencing allows to monitor genome wide the genomic regions occupied by a transcription factors or a specific histone mark. The technology is improving and allows to interrogating only small cell populations15. It thus opens new avenues in the dynamics of specific epigenetic landscapes during embryonic development.

The heart is the first organ to be formed and to become functional in the embryo. The heart is built from many cell lineages that arise from the first and second embryonic heart fields1. From the post-fertilization blastocyst stage up to the shaped heart, embryonic cells have thus to make many cell fate decisions. Gene transcription is regulated in a time- and space-dependent manner and is a key biological process that underlies cell fate decision during embryonic development. Such a process is mediated by specific transcription factors which bind regulatory regions within the genome including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to modifications such as acetylation, methylation, ubiquitinylation, and/or phosphorylation. Histone modification leads to repressed, activated or poised gene transcription depending upon which lysine residue of histone is modified2.
Chromatin immunoprecipitation assay (ChIP) has been set up years ago3 and is currently the most broadly used technology in order to identify targets of either modified histones or transcription factors4. Following immunoprecipitation of histones or transcription factors, bound DNA can be either amplified by polymerase chain reaction (PCR) or sequenced. ChIP has technically overcome more challenging gel retardation assays5. However ChIP does not imply direct binding of a transcription factor to DNA, an advantage of gel retardation assay. On the other hand, ChIP combined to DNA sequencing has opened a new genome-wide perspective on gene regulation.
ES cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development6 and provide in principle enough material for ChIP. Furthermore, human ES cells represent a human cell model of cardiogenesis although their cardiogenic potential depends upon their epigenetic signature7. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. However, the genome is transcribed in a time- and cell type-specific manner8. Epigenetic regulation of gene transcription has to be studied within localized regions. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

<table><tbody><tr><td>Formaldehyde&amp;nbsp;</td><td>Sigma</td><td>F8775</td><td>Cell Fixation&amp;nbsp;</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td>Cross-link stop</td></tr><tr><td>Aprotinin</td><td>Fluka</td><td>10820</td><td>Proteases inhibitor</td></tr><tr><td>Leupeptin hemisulfate</td><td>Sigma</td><td>L2882</td><td>Proteases inhibitor</td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td>Proteases inhibitor</td></tr><tr><td>Protein A magnetic beads&amp;nbsp;</td><td>Life technologies</td><td>10001D</td><td>Immunoprecipitation</td></tr><tr><td>SPRI magnetic beads</td><td>Thermo Scientific</td><td>15002-01</td><td>DNA purification</td></tr><tr><td>Proteinase K</td><td>Life technologies</td><td>25530-015</td><td>Protein digestion&amp;nbsp;</td></tr><tr><td>DNA BR standard&amp;nbsp;</td><td>Life technologies</td><td>Q32850</td><td>Calibration range&amp;nbsp;</td></tr><tr><td>Syber green</td><td>Molecular Probes</td><td>S-11484</td><td>DNA quantification</td></tr><tr><td>TE buffer&amp;nbsp;</td><td>Invitrogen</td><td>P7589</td><td>DNA quantification</td></tr><tr><td>PBX 1x</td><td>Life technologies</td><td>14190-094</td><td>Washing</td></tr><tr><td>DNase RNase free water</td><td>Life technologies</td><td>10977-035</td><td>Dilution</td></tr><tr><td>Axygen tube</td><td>Axygen</td><td>MCT-175-C</td><td>ChIP purifiction</td></tr><tr><td>&lt;strong&gt;Antibody&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Reference&lt;/strong&gt;</td><td>&lt;strong&gt;ChIP concentration&lt;/strong&gt;</td></tr><tr><td>H3K27ac</td><td>Abcam</td><td>ab4729</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr><tr><td>H3K4me1</td><td>Diagenode</td><td>C15410194 (pAb-194-050)</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr><tr><td>H3K36me3</td><td>Diagenode</td><td>C15410058 (pAb-058-050)</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr><tr><td>H3K9me2</td><td>Diagenode</td><td>C15410060 (pAb-060-050)</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr><tr><td>H3K4me3</td><td>Diagenode</td><td>C15410030 (pAb-030-050)</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr><tr><td>H3K27me3</td><td>Diagenode</td><td>C15410069 (pAb-069-050)</td><td>3 &amp;micro;g for ESC and EBs, 1 &amp;micro;g for tissues</td></tr></tbody></table>

epigenetic regulation of cardiac differentiation of embryonic stem cells and tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

Figure 1A illustrates first the preparation of DNA-binding beads and quality control using DNA of different sizes (1 kb ladder). 0ne, 2 and 2.5 volumes (1 to 3) of beads was added to one volume of the sample to purify high and low molecular size DNA fragments.
Figures 1 B,C,D are typical examples of DNA gels from whole sonicated DNA extracted from mouse ES cells, embryoid bodies or a cardiac embryonic region (pooled 25 E9.5 ventricles), respectively.
When ES cells differentiate toward a cardiac cell fate, they transit first through a mesendodermal and then a mesodermal state. Such a developmental journey includes a step by which epithelial cells have to undergo an epithelio-mesenchymal transition
Figure 2 shows PCR results following immunoprecipitation of modified histones. The experiment was designed to look at the epigenetic regulation of epithelio-mesenchymal transition genes in wild type or gene-mutated cells when ES cells differentiate toward a mesodermal cell fate. Chromatin from wild type or mutated ES cells treated with a morphogen like BMP2 was immunoprecipitated using anti H3K4me1, H3K36me3 and H3K9me2 antibodies. Binding of modified histones is monitored by PCR of genomic (enhancers, A, B, C, D, E) regions of cadherin and twist genes.
Differentiation genes feature bivalent domains in ES cells12. Their 5&#39; UTR regions close to their promoters are indeed occupied by both H3K4me3 a chromatin activating mark and H3K27me3, a repressed mark. These marks are modified when cells are challenged by morphogens such as BMP2 7. These marks are however variable depending upon the human ES cells lines likely because of the original blastocyst they derive from even when undifferentiated (Figure 3).
The protocols described above are suitable for ChIP sequencing even starting from a low amount of material; ChIP experiments were performed from chromatin extracted from AVC, OFT and ventricles of E9.5 mouse embryos in order to uncover novel genes and enhancers playing a role in defining the identity of these specific cardiac regions. Figure 4 shows 2 genomic regions of myocardial genes in the E9.5 mouse ventricle enriched by an acetylated H3K27 and of one pacemaker-specific (i.e., not ventricular) gene not enriched by acetylated H3K27.
<img alt="Figure 1" src="/files/ftp_upload/53874/53874fig1.jpg" /> 
Figure 1. Electrophoresis Data: DNA-binding Beads Checking and Sonication Validation. (A) DNA purified by DNA-binding beads, 1 volume of beads/1 volume of DNA (1) and 2 volumes of beads/1 volume of DNA (2) and 2.5 volume of beads/1 volume of DNA (3). Size of DNA fragments obtained after reverse crosslink of input fractions of mouse ES cells (B), embryoid bodies (C), and embryonic cardiac tissue (D). The samples were run on 2% agarose electrophoresis gels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53874/53874fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53874/53874fig2.jpg" /> 
Figure 2. ChIP Data Analysis. (A) Enrichment of H3K27ac epigenetic mark on E-cadherin enhancers/promoter (A&#160;- E&#160;regions). (B) Enrichment of H3K4me1, H3K36me3 and H3K9me2 epigenetic marks on Twist enhancers/promoter (A-C regions). Genomic regions are amplified by qPCR to measure the histone enrichment vs the input following ChIP. Results are means&#177;SEM from 3 experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53874/53874fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53874/53874fig3.jpg" /> 
Figure 3. Sequential ChIP. Chromatin was extracted from 4 human ES cell lines and ChIP was performed sequentially using anti H3K4me3 and then anti-H3K27me3 antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53874/53874fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53874/53874fig4.jpg" /> 
Figure 4. ChIP from Chromatin Extracted from Specific Embryonic Cardiac Regions. Chromatin was extracted from 25 ventricles of E9.5 mouse embryos (2 litters) lysed and sonicated. The anti-H3K27ac antibody was used for immunoprecipitation. The DNA was used for sequencing. The figure shows genomic regions of the cardiac genes Nkx2.5 and Tbx5 enriched in the modified histone and one genomic region of Shox2 pacemaker-specific gene not enriched and not expressed in the ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53874/53874fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues at JoVE.com

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

epigenetic-regulation-cardiac-differentiation-embryonic-stem-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.0K Views.  Université Aix-Marseille. A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Video: Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

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.

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 ...

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 ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

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 ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biology

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 ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Summary

Explore More Videos

Chapters in this video

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

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

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

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

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

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