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>
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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. 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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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

Our method aims to generate heart field-specific cardiac progenitor cells from mouse embryonic stem cells. This heart field reporter stem cell line allows for the high-throughput study of the mechanisms underlying congenital heart disease, providing a system that is amenable to genetic and pharmacological manipulation. There are several chamber-specific congenital heart diseases.By recapitulating cardiogenesis in vitro, this protocol allows the study of disease mechanism and the development of future regenerative therapies. This protocol can provide insight into how cardiac progenitor cells of different chambers are specified during heart development. Begin by growing transgenic mouse embryonic stem cells in 0.1%gelatin-coated T25 flasks in 2i medium.When the cells reach 70 to 80%confluence, rinse the cultures with PBS and add one milliliter of trypsin per flask to dissociate the culture into single cells at 37 degrees Celsius for three minutes. When the cells have detached, neutralize the reaction with four milliliters 10 FBS in DMEM and count the cells by an appropriate method. Dilute the cells to an approximately three times 10 to the five cells per fresh medium concentration, and collect the cells by centrifugation.Then resuspend the pellet in five milliliters of fresh 2i medium for replating onto new 0.1%gelatin-coated T25 flasks. For CPC generation from cardiac spheroids, collect 2.5 times 10 to the six of the detached transgenic mouse embryonic stem cell sample by centrifugation, and resuspend the cells in 25 milliliters of SFD medium. Plate the cell suspension into one 150-by-25-millimeter sterile plate for incubation at 37 degrees Celsius and 5%carbon dioxide for 48 hours.At the end of the incubation, collect the cardiac spheroids into a conical tube. Sediment the spheroids by centrifugation to facilitate the selective isolation of the spheroids and to avoid single cells. Resuspend the spheroids in 25 milliliters of fresh SFD medium supplemented with one nanogram per milliliter of activin A and 1.5 nanograms per milliliter of bone morphogenetic protein four.Replate the spheroids back onto the same culture plate for a 24-hour incubation in the cell culture incubator. The next day, recollect all of the cardiac spheroids by centrifugation. Resuspend the differentiated embryoid bodies in 25 milliliters of fresh SFD medium for plating in an ultra-low attachment, 75-centimeter squared flask in the cell culture incubator for 48 hours.For isolation of heart field-specific CPC using fluorescent reporters, collect the embryoid bodies by centrifugation and dissociate the 3D cultures with one milliliter of trypsin at 37 degrees Celsius for three minutes. At the end of the incubation, mix well by pipetting to dissociate the cells and neutralize the reaction with four milliliters of 10%FBS in DMEM. Pass the mixture over a 70-micrometer strainer to remove any non-dissociated embryoid bodies, and sediment the filtered cells by centrifugation.To sort the CPCs by their fluorescent reporter expression, resuspend the pellet in 500 microliters FACS solution and filter the cells through a 40-micrometer cell strainer into a five-milliliter polystyrene round-bottom tube on ice. Then sort the cells to isolate the RFP-and GFP-expressing cells by FACS, collecting the cells in one milliliter of FBS. To isolate first versus second heart field CPCs based on their surface protein Cxcr4 expression, resuspend single mouse embryonic stem cell RFP-expressing cardiac progenitor cells in 300 microliters of 10%FBS in PBS supplemented with fluorescence-conjugated anti-Cxcr4 antibody.After five minutes at room temperature, wash the cells three times in one to two milliliters of fresh, cold PBS per wash. After the last wash, resuspend the cells in 500 microliters of FACS solution and filter the cells through a 40-micrometer strainer into a five-milliliter, round-bottom tube. Then sort the cells by their Cxcr4 expression, collecting both the Cxcr4-positive and negative populations into individual tubes containing one milliliter of FPS per tube on ice.To re-culture the FACS-isolated, heart field-specific cardiac progenitor cells, collect the sorted cells by centrifugation and resuspend the pellets in SFD medium. Then seed approximately three times 10 to the four cells per well in a 0.1%gelatin-coated, 384-well plate. If increased cell death is noted after sorting, add 10-micromolar ROCK inhibitor to each well.After two days of culture, spontaneous beating should be observed. To analyze the ability of plated CPCs to differentiate to cardiomyocytes, collect the cells at day 12 of differentiation with trypsin, as demonstrated, to isolate the single cardiomyocytes and resuspend the cells in 4%paraformaldehyde. After 30 minutes at room temperature, collect the fixed cells by centrifugation and wash the pellet in PBS to remove the excess fixative.Next, resuspend the cells in 10%FBS in PBS, and incubate half of the cell sample with mouse anti-troponin T antibody and use the other half of the sample as a negative control. After 30 minutes at room temperature, wash the cells two times in fresh PBS and resuspend the samples in 10%FBS plus PBS supplemented with an appropriate secondary antibody. After another 30-minute incubation at room temperature, wash the cells two times in fresh PBS per wash and resuspend the cells in 200 microliters of PBS per tube for analysis on a flow cytometer.After approximately 132 hours of differentiation, Tbx1-RFP and Hcn4-GFP cardiac progenitor cells can be detected by fluorescence microscopy. Generally, both GFP and RFP cells appear approximately around the same time, and the two populations of progenitor cells continue to expand in close proximity and typically in a complementary pattern. Adjusting the concentrations of activin A and bone morphogenetic protein four will alter the percentages of first versus second heart field cardiac progenitor cells.Similarly, using an RFP reporter mouse embryonic stem cell line, after 132 hours of differentiation, RFP-positive cardiac progenitor cells appear. After immunostaining for Cxcr4, RFP-positive, Cxcr4-positive, and RFP-positive, Cxcr4-negative cells can be isolated. Immunostaining for cardiac troponin T at day 12 of differentiation confirms that first heart field cells differentiate mainly into myocytes.Similarly, cells derived from RFP-positive, Cxcr4-negative cardiac progenitor cells give rise to cardiomyocytes at much higher percentages compared to Cxcr4 single positive cardiac progenitor cells. Occasionally, mouse embryonic stem cells fail to differentiate efficiently and form very low numbers of heart field-specific cardiac progenitor cells. The timing, concentration, and consistency of the addition of the cytokines and the number of the cells are critical for a successful generation of chamber-specific cardiac progenitor cells.Following this procedure, investigators can analyze the physiological properties of the different populations of cardiac progenitor cells to obtain further insight into their specification and function.

in-vitro-generation-of-heart-field-specific-cardiac-progenitor-cells

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JoVE Journal

Developmental Biology

6.2K visualizações.  Johns Hopkins School of Medicine. Nosso método visa gerar células progenitoras cardíacas específicas do campo cardíaco a partir de células-tronco embrionárias de camundongos. Esta linha de células-tronco do campo cardíaco permite o estudo de alto rendimento dos mecanismos subjacentes à doença cardíaca congênita, fornecendo um sistema que é favorável à manipulação genética e farmacológica. Existem várias doenças cardíacas congênitas específicas da câmara.Ao recapitular a cardiogênese in vitro,...