The authors greatly appreciate Dr. Lingli Wang for her help with the animal protocol, and the Department of Pediatrics at Stanford for infrastructure support. This work was supported by NIH/NHLBI K99 HL135258 (to M.G.).

All animal procedures were approved by the Administrative Panel on Laboratory Animal Care (APLAC31608) at Stanford University.
1. Preparation of buffers
<ol>
	<li>Prepare the digestion buffer using the reagents listed in Table 1.
	<ol>
		<li>Prepare the DNase I stock solution: Dissolve DNase I in RNase-free water for a stock solution of 2,000 units/mL (1 mg/mL). Aliquot and store the stock solution at -20 &#730;C.</li>
		<li>Prepare the liberase solution: Dissolve liberase with RNase-free water for a stock solution of 5 mg/mL. Rotate it on a roller bank at 4 &#730;C for 30 min. Aliquot and store the stock solution at -20 &#730;C.</li>
	</ol>
	</li>
	<li>Prepare the sorting buffer by diluting 0.5 M ethylenediaminetetraacetic acid (EDTA) and 10% bovine serum albumin (BSA) stock solution with 1x phosphate buffered saline (PBS) for a final concentration of 2 mM of EDTA and 0.5% of BSA.</li>
</ol>
2. Heart collection
NOTE: Six Sprague Dawley rats weighing 50&#8211;100 g were used in this protocol. No gender difference was observed.
<ol>
	<li>Euthanize the rat with CO2 and place it in a supine position on a dissecting platform. Pin the extremities down and sterilize both the abdomen and chest with 70% ethanol.</li>
	<li>Open the abdomen with scissors and insert a 21 G needle into the portion of the posterior vena cava located behind the intestines. Pull the syringe plunger back, withdrawing the blood from the vein until the liver appears lighter in color and no more blood can be withdrawn, indicating sufficient blood removal.</li>
	<li>Using scissors, open the chest carefully to avoid damaging the lung and the heart. Lift the aorta arch/atrium with the forceps and cut the aorta, pulmonary artery, pulmonary veins, and vena cava to liberate and remove the heart.</li>
	<li>Wash the heart with 50 mL of cold 1x Hanks&#39; balanced salt solution (HBSS) buffer 3x to remove excessive blood.</li>
	<li>Under a microdissection microscope, remove the right ventricular free wall into a 5 mL tube containing Dulbecco&#39;s modified Eagle medium (DMEM) (Figure 1A,B).
	<ol>
		<li>Lay the heart on its flatter, posterior face to identify the left and right side.</li>
		<li>Locate the pulmonary artery, the most anterior of the veins and arteries branching from the top of the heart and cut through the pulmonary artery down to the right ventricular chamber. On the anterior face of the heart, cut along the septum until reaching the apex. Continue cutting from the apex up the posterior side of the heart along the septum until the pulmonary artery and right ventricular chamber junction point (Figure 1A).</li>
		<li>Starting from the pulmonary artery and right ventricular junction point, cut both the anterior and posterior side of the heart perpendicularly to the previous dissection and away from the septum, until the right ventricular free wall is liberated from the rest of the heart (Figure 1B).</li>
	</ol>
	</li>
	<li>Under the microdissection microscope, remove the left ventricular free wall into a 5 mL tube containing DMEM (Figure 1C,D).
	<ol>
		<li>Locate the aorta, branching from the top of the heart behind the pulmonary artery, and repeat the method described in step 2.5 to cut off the left ventricular free wall.</li>
	</ol>
	</li>
</ol>
3. EEC digestion
<ol>
	<li>Place both right and left ventricular free wall tissues in a 60 cm culture dish with the inner surface lying flat, facing down (Figure 2). 
	NOTE: The inner surface is the interior face of the slightly concave shaped ventricle as shown in Figure 2A,C.</li>
	<li>Add 0.5&#8211;1 mL of digestion buffer to the dish, placing the tip of the pipette directly under the tissue. Continue until only the inner surface is immersed.</li>
	<li>Incubate the dish in a 5% CO2, 37 &#730;C incubator for 5 min.</li>
	<li>Add EC medium to the culture dish to stop the digestion. 
	NOTE: Keep the amount of digestion buffer to EC medium as a 1:5 ratio.</li>
	<li>Use a 1 mL pipette to flush the inner surface of the ventricles with EC medium and transfer the runoff into a 50 mL collection tube through a 40 mm strainer (Figure 2E). Keep the collections on ice for downstream purification.</li>
</ol>
4. CEC digestion
<ol>
	<li>Cut along the outer surface of the left ventricle without contamination from the inner layer (Figure 2B,D) and place each in a separate 5 mL tube containing 1 mL of the digestion buffer. 
	NOTE: The left ventricle can be identified as the thicker ventricle, and the outer surface can be determined as the exterior face of the slightly concave ventricle.</li>
	<li>Using dissection scissors, mince the ventricular wall into small, 1 mm3 pieces.</li>
	<li>Incubate the tube in a 37 &#730;C water bath for about 15&#8211;20 min. Vortex every 2&#8211;3 min.</li>
	<li>Pipette 4 mL of EC medium into the 5 mL tube to terminate the digestion.</li>
	<li>Transfer the solution with a 5 mL serological pipette into a 50 mL tube through a 40 mm strainer (Figure 2E). Keep the collections on ice for downstream purification.</li>
</ol>
5. Cell collection
<ol>
	<li>Centrifuge the tubes at 300 x g for 10 min at room temperature (RT) and then aspirate the supernatant.</li>
	<li>If the pellet appears red, resuspend the pellet in 1&#8211;2 mL of 1x red blood cell (RBC) lysing buffer (Figure 2F). 
	NOTE: If there are no RBCs, proceed to step 6.</li>
	<li>Incubate the tubes in a 37 &#730;C water bath for 5 min.</li>
	<li>Pipette 10 mL of PBS into the 50 mL tubes to stop the lysis.</li>
	<li>Centrifuge the tubes at 300 x g for 10 min at RT, and then aspirate the supernatant.</li>
</ol>
6. EC sorting
<ol>
	<li>Add 90 &#956;L of sorting buffer and 10 &#956;L of anti-CD31 PE antibody into the 50 mL tubes.</li>
	<li>Vortex the tubes and then incubate them for 10 min in a 4 &#730;C fridge.</li>
	<li>Add 10 mL of sorting buffer into the tubes, mix thoroughly, and then centrifuge at 300 x g for 10 min at RT.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 80 &#181;L of sorting buffer and 20 &#181;L of anti-PE microbeads.</li>
	<li>Vortex the tubes and incubate at 4 &#730;C for 15 min.</li>
	<li>Wash the cells by adding 10 mL of sorting buffer into the tubes, mix thoroughly, and then centrifuge them at 300 x g for 10 min at RT.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 500 &#956;L of sorting buffer.</li>
	<li>Place a column containing superparamagnetic spheres into a magnetic separator and rinse the column with 3 mL of sorting buffer.</li>
	<li>After the column are &#8220;flow stop&#8221;, pipette 500 &#956;L of the cell suspension into the columns and then wash the columns with sorting buffer. Repeat the wash 3x, each with 3 mL of sorting buffer (Figure 2G).</li>
	<li>Remove the columns from the separator and place them on top of a new 15 mL collection tube.</li>
	<li>With a pipette, add 5 mL of EC medium to the columns, and propel the solution through using a column plunger.</li>
	<li>Centrifuge the collection tubes at 300 x g for 10 min at RT and then aspirate the supernatant.</li>
	<li>Extract the RNA from the cell pellet of the EEC and CEC samples using a kit, following the manufacturer&#8217;s instructions. A minimum of 500 ng of RNA can be obtained. 
	NOTE: After RNA extraction, the sample can be stored at -80 &#730;C.</li>
	<li>Reverse transcribe 500 ng of RNA to get cDNA using a random primer and a kit, following the manufacturer&#8217;s instructions.</li>
	<li>Perform quantitative PCR for the validation of endocardial and endothelial populations (Figure 2H). Primer sequences are listed in Table 2.
	<ol>
		<li>Add 5 &#956;L of EEC or CEC cDNA (1 ng/&#956;L) into the wells of a 384 qPCR plate. Add the EEC or CEC cDNA into enough wells so that there are three wells per number of primers.</li>
		<li>Mix 6 &#956;L of 2x qPCR SYBR green buffer with 0.5 &#956;L of each 10 &#956;M primer, including both forward and reverse primers, and add them into a single well corresponding to each candidate gene.</li>
		<li>Seal the plate with plastic film and centrifuge for 1 min at 300 x g at RT.</li>
		<li>Place the plate into a qPCR machine and then start the program at 95 &#730;C for 3 min, followed by 95 &#730;C for 15 s, and then 55 &#730;C for 60 s. Repeat the subsequent two steps for 45 cycles.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gladka, M. 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=Single-Cell+Sequencing+of+the+Healthy+and+Diseased+Heart+Reveals+Cytoskeleton-Associated+Protein+4+as+a+New+Modulator+of+Fibroblasts+Activation.">Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation.</a> Circulation. 138 (2), 166-180 (2018).</li><li>Koren, C. W., Sveinbjornsson, B., Smedsrod, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+endocardial+endothelial+cells+from+Atlantic+salmon+(Salmo+salar)+and+Atlantic+cod+(Gadus+morhua).">Isolation and culture of endocardial endothelial cells from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua).</a> Cell and Tissue Research. 290 (1), 89-99 (1997).</li><li>Tian, 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=Subepicardial+endothelial+cells+invade+the+embryonic+ventricle+wall+to+form+coronary+arteries.">Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries.</a> Cell Research. 23 (9), 1075-1090 (2013).</li><li>Wu, 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=Endocardial+cells+form+the+coronary+arteries+by+angiogenesis+through+myocardial-endocardial+VEGF+signaling.">Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.</a> Cell. 151 (5), 1083-1096 (2012).</li><li>Li, Y., Lui, K. O., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessing+endothelial-to-mesenchymal+transition+in+cardiovascular+diseases.">Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases.</a> Nature Reviews in Cardiology. 15 (8), 445-456 (2018).</li><li>Zhang, H., Lui, K. O., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+Cell+Plasticity+in+Cardiac+Development,+Diseases+and+Regeneration.">Endocardial Cell Plasticity in Cardiac Development, Diseases and Regeneration.</a> Circulation Research. 122 (5), 774-789 (2018).</li><li>Yu, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+coronary+artery-derived+endothelial+cells+in+vivo+from+patients+undergoing+percutaneous+coronary+interventions.">Isolation and characterization of human coronary artery-derived endothelial cells in vivo from patients undergoing percutaneous coronary interventions.</a> Journal of Vascular Research. 46 (5), 487-494 (2009).</li><li>Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. 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The authors have nothing to disclose.

CECs and EECs differ in origin, markers, and functions, and thus could play unique roles in development and disease. Existing protocols for endothelial cell isolation are limited to macrovascular tissues, neglecting the collection of EECs, thus restricting the study of CEC- and EEC-specific functions. It is essential to isolate and study these two populations independently, as this knowledge would provide a reference for the differentiation of iPSCs into CECs and EECs for future studies and facilitate the examination of these cell populations for potential therapeutic targets for various cardiac diseases. This novel protocol outlines a method for the isolation of EECs from the inner surface and CECs from the outer surface of the ventricular free wall of adult rats.
It is critical to control the timing for each step very precisely in this protocol. Because the number of EECs is very limited in rat heart tissue, we minimized the digestion time of the inner layer of the heart to prevent cellular damage and more importantly, CEC contamination. Immediate addition of the EC medium solution to terminate the enzymatic reaction is also very important for maintaining high cell viability. A red pellet following cell collection suggests the presence of a large number of RBCs. Depending on the amount of RBC contamination, 1&#8211;2 mL of RBC lysis buffer can be added to degrade the RBCs. Incubation must be carefully timed to avoid significant damage to the cells, and PBS is promptly added to terminate the reaction. Using the current protocol, we were able to isolate 105 EECs from six rat hearts. These cells could be seeded onto a cell culture dish for further expansion and characterization.
When preparing the tissue for free wall collection, the heart was washed with HBSS to remove the majority of the remaining RBCs. HBSS is the recommended medium due to its clear appearance, which enables the visualization of blood cells, in contrast to DMED containing phenol red. The composition of the digestion buffer ensures sufficient liberation of endothelial cells, where liberase digestion enzyme strips the tissue of the exposed cells, DNase I eliminates DNA from the dead cells to promote cell detachment that may be inhibited by DNA&#8217;s adhesive quality, HEPES buffer balances the pH, and DMEM is a modified basal medium with a higher concentration of amino acids and vitamins for better cell maintenance and contains the calcium necessary to activate the liberase.
According to the single cell RNA sequencing (scRNA-seq) obtained from both human15 and mouse heart10, several markers attributed to specific EC populations were reported. We selected some of the most enriched genes to examine the purity of isolated EECs (i.e., Npr3, Cdh11, and Hapln1) and EECs (i.e., Mgll, Fabp4, and Cd36). Relative to &#946;-Actin, Npr3, Cdh11, and Hapln1 markers demonstrated increased expression in EECs compared to CECs. Similarly, the expression of Mgll, Fabp4, and Cd36 markers was greater in CECs compared to EECs. The uniquely expressed markers for each sample is in accord with markers characteristic to EECs and CECs respectively, indicating successful isolation.
However, the current protocol cannot rule out cross contamination between the two EC populations during the isolation, even with carefully controlled sequential digestions. Therefore, some cell surface markers can be applied for further purification. For example, NPR3 generally labels the endocardium10, whereas APJ can trace a majority of the CECs16. Because these two markers are expressed on the cell surface, they could be used for fluorescence activated cell sorting (FACS), and antibodies used to further purify distinct EC populations. In addition, human15 and mouse10 heart scRNA-seq can confirm enrichment of Npr3 in EECs, and Cd36 can be potentially used for CEC purification.
In conclusion, the presented protocol outlines the independent isolation of EECs and CECs from the rat heart. The comprehensive identification of cellular properties, enabled by cell isolation, can be utilized for significant downstream applications.

This article provides a detailed protocol (modified from Gladka et al.1) for the dissection and subsequent isolation of endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) from rat hearts. The ability to investigate these cell populations independently would enable the exploration of cell type-specific mechanisms underlying a variety of heart diseases that could serve as potential therapeutic targets. A successful method for the collection of these cell populations independently has yet to be published, however.
CECs differ from EECs in regards to their origin, markers, and functions during heart development and disease1,2,3,4,5,6,7. EECs stem from the ventral surface of the cardiac mesoderm3. They arise from Flk1+ progenitor cells in response to VEGF and HIF signaling and form the innermost layer of the three discrete regions of the developing heart: the atrium, ventricle, and sinus venosus3,6. Genetic lineage tracing suggests that the pluripotent endocardial cells of the sinus venosus derive venous cells, which migrate to form the subepicardial layer3. Subsequently, the subepicardial layer differentiates into coronary arteries and veins, including CECs, which remain across the peripheral ventricular free wall3,4. This endocardial to endothelial pathway is regulated by VEGFC, ELA/APJ, and SOX17 signaling3,4,6,8,9. The ventricular endocardium derives the fewer CECs of the interventricular septum by an unknown mechanism3. Subsequently, localized differentiation between EECs and CECs is suggested by markers specific to these two cell populations, including Mgll, Fabp4, and Cd36 expression in CECs, or Npr3, Cdh11, and Hapln1 expression in EECs3,5,10.
EECs and CECs play different roles in cardiac function. Endocardial to mesenchymal transition, valve formation, chamber maturation, outflow tract regulation, and atrioventricular canal development are contingent on EECs6. Alternatively, CECs contribute to vasomotor tone and inflammation of coronary arteries11. These variances in function result in individualized roles in disease development4,12. For instance, evidence suggests that malfunctioning EECs may lead to congenital valve disease6, noncompaction myocardium6, atrioventricular septal effect6, endocardial fibroelastosis13, hypoplastic left heart syndrome13, ventricular hypoplasia13, and cardiac hypertrophy12. Similarly, studies have found that abnormal CECs contribute to coronary artery disease14 and thrombosis11.
Successful isolation of EECs and CECs is necessary to achieve comprehensive knowledge of these two cell populations, which could be utilized in both the research and clinical settings. Determining the growth and differentiation factors of these cell populations would provide a reference for the differentiation of endothelial subtypes from induced pluripotent stem cells (iPSCs). Further, complete identification of the variances in the development, regulation, and function of EECs and CECs is vital for understanding the genomic and epigenomic factors responsible for numerous heart diseases in a cell type-specific manner. This article outlines steps for the successful collection of EECs and CECs independently, and provides evidence of separation by assessing the gene expression levels of cell type-specific markers.

<table>
	<tbody>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>15ml Tubes</td>
			<td>Eppendorf</td>
			<td>0030122151</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Tubes</td>
			<td>Nunc</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml Tubes</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 PE</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-505</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>CFX384 Touch Real-Time PCR Detection System</td>
			<td>Bio-rad</td>
			<td>1855485</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LK 003172</td>
			<td>1 mg/mL in water</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium-2 (EGM-2)</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EC Medium</td>
		</tr>
		<tr>
			<td>Ethylendiaminetetraacetic Acid (EDTA)</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td>0.5 M</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS) X1</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-Shell 384-Well PCR Plates, thin wall, skirted, clear/white</td>
			<td>Bio-rad</td>
			<td>HSP3805</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM</td>
			<td>Sigma Aldrich</td>
			<td>5401119001</td>
			<td>5 mg/mL</td>
		</tr>
		<tr>
			<td>MACS LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>For EC isolation</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>For EC isolation</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-rad</td>
			<td>MSB1001</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>F.S.T</td>
			<td>11002-12</td>
			<td>For heart harvest</td>
		</tr>
		<tr>
			<td>Nylon Sterile Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td>40 mm</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) X1</td>
			<td>Gibco</td>
			<td>1001023</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>A25780</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Quick-RNA Microprep Kit</td>
			<td>Zymo Research</td>
			<td>R1050</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps - SuperGrip Tips</td>
			<td>F.S.T</td>
			<td>00632-11</td>
			<td>For heart harvest</td>
		</tr>
		<tr>
			<td>Strabismus Scissors - Tungsten Carbide</td>
			<td>F.S.T</td>
			<td>14574-11</td>
			<td>For heart harvest</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - Microserrated</td>
			<td>F.S.T</td>
			<td>15007-08</td>
			<td>For heart harvest</td>
		</tr>
	</tbody>
</table>

isolation of endocardial and coronary endothelial cells from the ventricular free wall of the rat heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

The process of EEC and CEC isolation is described in Figure 2. The successful isolation of EECs and CECs was determined by assessing the presence of pan-endothelial cell markers, as well as those distinct to the two subtype populations. As predicted, qPCR revealed that relative to &#946;-actin, EECs expressed higher levels of endocardial markers Npr3, Hapln1, and Cdh11 compared to CECs (Figure 3A). Likewise, CECs expressed higher levels of coronary markers Fabp4, Mgll, and Cd36 compared to EECs (Figure 3B). Additionally, both EECs and CECs expressed the pan-EC marker gene Cdh5, with slightly higher levels in CEC (Figure 3C).
 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61126/61126fig1v5.jpg" /> 
Figure 1: Diagram of heart dissection.&#160;(A) First cut made to separate the right ventricle free wall from the septum. (B) Second cut made to liberate the right ventricle completely. (C) First cut made to separate the left ventricle free wall from the septum. (D) Second cut made to liberate the left ventricle completely. <a href="https://www.jove.com/files/ftp_upload/61126/61126fig1v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61126/61126fig2v5.jpg" /> 
Figure 2: Diagram of digestion set-up of the CECs and EECs and the following arrangement for cell sorting.&#160;(A) Innermost free ventricular wall and (B) outermost ventricular free wall were (C) immersed in digestion buffer or (D) digested in digestion buffer respectively. (E) Collection and filtration of cell solutions followed by (F) red blood cell (RBC) lysis and (G) magnetic-activated cell sorting (MACS) using the CD31 antibody. (h) Purified ECs were processed for gene expression verification using qPCR. <a href="https://www.jove.com/files/ftp_upload/61126/61126fig2v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61126/61126fig3v5.jpg" /> 
Figure 3: qPCR data verifying isolation of CECs and EECs.&#160;(A) Gene expression levels of the EEC markers Npr3, Cdh11, and Hapln1 in EECs and CECs were quantified by real-time PCR. (B) Gene expression levels of the CEC markers Mgll, Fabp4, and Cd36 in EECs and CECs were quantified by real-time PCR. (C) The pan-EC marker Cdh5 was quantified in EECs and CECs by real-time PCR. (n = 3 in each group). Bars represent mean &#177; SEM. *p &#60; 0.05, **p &#60; 0.01 vs. EEC, unpaired t-test. <a href="https://www.jove.com/files/ftp_upload/61126/61126fig3v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Table 1. Digestion Buffer (1.5ml)</td>
		</tr>
		<tr>
			<td>&#160;</td>
			<td>Stock Con.</td>
			<td>Final Con.</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Liberase TM</td>
			<td>5 mg/ml</td>
			<td>0.5 mg/ml</td>
			<td>150 ul</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>1 mg/ml</td>
			<td>20 ug/ml</td>
			<td>30 ul</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td>15 ul</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>-</td>
			<td>-</td>
			<td>1305 ul</td>
		</tr>
	</tbody>
</table>
Table 1: Recipe for the digestion buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Table 2. Primer Sequences</td>
		</tr>
		<tr>
			<td>Gene Name</td>
			<td>Forward</td>
			<td>Reverse</td>
		</tr>
		<tr>
			<td>Npr3</td>
			<td>TCCTTGCAAATCATGTGGCCTA</td>
			<td>GGAATCTTCCCGCAGCTCTC</td>
		</tr>
		<tr>
			<td>Cdh11</td>
			<td>GTGAATGGGACTGGGACTGG</td>
			<td>GTAATTTCTGGGGCCCTTGC</td>
		</tr>
		<tr>
			<td>Hapln1</td>
			<td>CCAGCTAAGTGGGACTCGAAG</td>
			<td>GGGCCATTTTCAGCTTGGATG</td>
		</tr>
		<tr>
			<td>Mgll</td>
			<td>CCCGGGGCCCAAAGAC</td>
			<td>GAAGATGAGGGCCTTGGGTG</td>
		</tr>
		<tr>
			<td>Fabp4</td>
			<td>AGAAGTGGGAGTTGGCTTCG</td>
			<td>ACTCTCTGACCGGATGACGA</td>
		</tr>
		<tr>
			<td>Cd36</td>
			<td>GCAAAACGACTGCAGGTCAA</td>
			<td>CCCGGTCACTTGGTTTCTGA</td>
		</tr>
		<tr>
			<td>Cdh5</td>
			<td>CCATTGAGACAGACCCCGAC</td>
			<td>TGTGGAACGTGTACTGCTGG</td>
		</tr>
		<tr>
			<td>B-actin</td>
			<td>TCTGTGTGGATTGGTGGCTC</td>
			<td>CGGACTCATCGTACTCCTGC</td>
		</tr>
	</tbody>
</table>
Table 2: Primer Sequences

Watch this Scientific Journal Video about Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart at JoVE.com

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

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Developmental Biology

16.8K Views.  Stanford School of Medicine. We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

Video: Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...