We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

NOTE: All experiments were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.
1. Excise the Aortic Valve
<ol>
	<li>Using an approved euthanasia technique for the embryonic stage of interest, euthanize a pregnant mouse with pups at the desired embryonic day (E) of development.</li>
	<li>Use 70% ethanol to sterilize the abdomen of the pregnant female. Lift the skin and muscle of the lower abdomen up and away from the internal organs, and dissect open the female&#8217;s lower abdominal cavity to allow access to the uterine horn.</li>
	<li>To retrieve the uterine horn, hold the cervix with forceps and carefully cut caudal to the forceps. Lift the uterine horn, cutting any connective tissue that keeps the horn in place, and complete the removal by cutting the junction of the uterine horn with the oviduct.</li>
	<li>Place the dissected uterine horn in a Petri dish containing cold 0.1 M Tris buffer (pH 7.6) and rinse as needed. Then, cut the uterine horn open length-wise to expose the embryonic sacs.</li>
	<li>To expose the embryo, cut open an embryonic sac at the junction of the placenta and the embryo and cut the umbilical vessels to free the embryo. Place the dissected embryo in a second Petri dish with cold 0.1 M Tris buffer. Return the first Petri dish with the remaining undissected embryos to ice until ready for the next embryo.</li>
	<li>To improve chest wall access within the embryo, decapitate the embryo. If genotyping is necessary, remove and save an extra tissue piece in an Eppendorf tube placed on ice.</li>
	<li>Place the embryo on its back, and cut the chest wall vertically along the side of the rib cage, near a forelimb, and horizontally above the diaphragm to visualize the heart. Then, open the chest wall to expose the heart.</li>
	<li>Using forceps to hold high along the great vessels, lift the heart using forceps and cut the vessels below. Then, cut above the forceps to free the heart. If the pulmonary vessels remain uncut, the lungs may be removed with the heart and should be removed before continuing.</li>
	<li>Cut and remove the pulmonary artery above the level of the valve; at the embryonic stages described herein, the pulmonary artery is slightly opaque, which aids in its discrimination from the valve region. Cut just below the pulmonary valve, avoiding the trabeculated ventricular myocardium and collect as described in step 1.10 if this region is also of interest. Here and in the next step, use capillary action to draw excess Tris buffer away from the region of interest prior to collecting in lysis buffer.</li>
	<li>Carefully remove the aorta distal to the aortic valve, just above the level of the valve; like the pulmonary artery, the aorta remains slightly opaque at these embryonic stages, aiding in its removal. Cut just below the aortic valve, again avoiding the trabeculated ventricular myocardium, and transfer the valve using forceps to an Eppendorf tube with 2 &#956;l lysis buffer (described in the Materials table) containing protease and phosphatase inhibitors on ice.</li>
	<li>Repeat steps 1.6-1.10 until the valves have been excised from all embryos, collecting each valve in a separate Eppendorf tube. If all embryos have the same genotype, all valves may be collected in a single Eppendorf tube.</li>
	<li>If genotyping is to be performed to determine which valves to pool together, immediately place samples in lysis buffer at -80 &#176;C until further use. Samples can then be pooled after genotyping (step 2.1.1).</li>
</ol>
2. Protein Extraction
<ol>
	<li>Thaw collected tissues on ice and centrifuge at 13,100 x g for 1 min&#160;to collect the tissues in lysis buffer at the bottom of the tube.
	<ol>
		<li>If embryos were genotyped, use a pipette to gently collect and pool lysis buffer containing the valve regions from embryos with similar genotypes. To ensure a strong band, pooling 3-4 valve regions from E13.5 embryos per sample is recommended.</li>
		<li>If all embryos were of the same genotype and all valve regions were collected together in Step 1.10, lyse the entire sample, as described in Step 2.2.</li>
	</ol>
	</li>
	<li>Check the volume of the resulting pooled sample and add lysis buffer to a total volume of 40 &#956;l; then, disrupt and homogenize samples using 5 mm stainless steel beads in Eppendorf tubes. Operate the lyser for 2-4 min at 50 Hz, as per the manufacturer&#8217;s instructions. Spin tubes briefly, (~13,100 x g for 1 min), and transfer samples to new tubes, leaving behind the insoluble debris.</li>
	<li>Prepare and separate samples for SDS-PAGE and transfer for subsequent western blot analysis, following a protocol such as Eslami&#160;et al9. Blotting for a loading control protein is essential for protein quantification.</li>
</ol>

<ol>
	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+cartilage+and+bone-related+proteins+in+pediatric+and+adult+diseased+aortic+valves.">Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves.</a> J Mol Cell Cardiol. 50, 561-569 (2011).</li><li>Chakraborty, S., 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=Twist1+promotes+heart+valve+cell+proliferation+and+extracellular+matrix+gene+expression+during+development+in+vivo+and+is+expressed+in+human+diseased+aortic+valves.">Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.</a> Dev Biol. 347, 167-179 (2010).</li><li>Lee, M. P., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+directly+regulates+genes+that+promote+cell+proliferation+and+migration+in+developing+heart+valves.">Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.</a> PLoS One. 6 (e29758), (2011).</li><li>Peacock, J. D., Huk, D. J., Ediriweera, H. N., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9+transcriptionally+represses+Spp1+to+prevent+matrix+mineralization+in+maturing+heart+valves+and+chondrocytes.">Sox9 transcriptionally represses Spp1 to prevent matrix mineralization in maturing heart valves and chondrocytes.</a> PLoS One. 6 (e26769), (2011).</li><li>Chakraborty, S., Cheek, J., Sakthivel, B., Aronow, B. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+gene+expression+profiles+in+developing+heart+valves+and+osteoblast+progenitor+cells.">Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.</a> Physiol Genomics. 35, 75-85 (2008).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13, 227-232 (2012).</li><li>Meng, 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=Expression+of+functional+Toll-like+receptors+2+and+4+in+human+aortic+valve+interstitial+cells:+potential+roles+in+aortic+valve+inflammation+and+stenosis.">Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis.</a> Am J Physiol Cell Physiol. 294, 29-35 (2008).</li><li>Dyer, L. A., Wu, Y., Moser, M., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPER-induced+BMP+signaling+promotes+coronary+artery+remodeling.">BMPER-induced BMP signaling promotes coronary artery remodeling.</a> Developmental Biology. 386, 385-394 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Garside, V. C., Chang, A. C., Karsan, A., Hoodless, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinating+Notch,+BMP,+and+TGF-beta+signaling+during+heart+valve+development.">Co-ordinating Notch, BMP, and TGF-beta signaling during heart valve development.</a> Cell Mol Life Sci. 70, 2899-2917 (2013).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Development. 127, 1607-1616 (2000).</li><li>Scherptong, R. W., 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=Morphogenesis+of+outflow+tract+rotation+during+cardiac+development:+the+pulmonary+push+concept.">Morphogenesis of outflow tract rotation during cardiac development: the pulmonary push concept.</a> Dev Dyn. 241, 1413-1422 (2012).</li><li>Mohler, E. R., Adam 3rd, L. P., McClelland, P., Graham, L., Hathaway, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+osteopontin+in+calcified+human+aortic+valves.">Detection of osteopontin in calcified human aortic valves.</a> Arterioscler Thromb Vasc Biol. 17, 547-552 (1997).</li></ol>

The authors have nothing to disclose.

The ability to quantify protein levels in early embryonic mouse and chick cardiac valve regions provides an additional tool for understanding the critical cellular signaling events for valve development. Our protocol described herein does not differ greatly from standard protein isolation procedures. However, by modifying some key steps, we have successfully obtained phosphorylated proteins from extremely small sample sizes. To achieve this outcome, the following steps are of particular importance. To ensure that quality protein is obtained, it is crucial to keep samples chilled, whether on ice or by using ice-cold buffers, in the presence of protease and, if necessary, phosphatase inhibitors. Further, homogenization with a device that is designed to process small sample volumes is essential for adequately disrupting cell membranes. Even with these precautions, the obtained yield of protein may be too low to detect via the Bradford assay with a spectrophotometer or a protein quantification kit and still have sample available for subsequent analyses. We were able to determine that each aortic valve region contains approximately 1 &#956;g of protein by pooling 7 aortic valve regions and using the entire sample for quantification. Despite this limitation, sufficient protein is obtained to detect phosphorylated and non-phosphorylated proteins, though the lack of prior quantification makes a loading control essential. We specifically utilized &#946;-actin because it is a high abundance protein, which allowed us to detect it even after stripping the membrane numerous times; its molecular weight differed from our proteins of interest; and it is ubiquitously expressed. A trial western blot with different numbers of pooled samples is recommended to determine how many tissues must be pooled for either embryonic cardiac valve regions at other stages of development or for other small tissues of interest. In addition, we recommend pooling at least three tissue samples for each protein sample to account for variability in the size of the dissected tissues as well as in the length of time each embryo sat on ice prior to dissection. If the protein of interest cannot be detected via western blot even after pooling samples, we recommend reducing sample volume and increasing the homogenization time to ensure that all potential protein is available for detection. Additionally, protease inhibitors may also be added to ice-cold Tris buffer during dissections for unstable or easily degraded proteins. If the protein of interest is expressed at low levels, additional troubleshooting during the western blot portion of the experiment, such as adjusting the primary antibody incubation or the substrate detection method, may help with visualization.
If the evidence (e.g., immunohistochemistry or in situ hybridization) suggests that a protein of interest is expressed both in the cardiac valves and the surrounding myocardium, the aortic or pulmonary artery myocardium can be teased apart gently with tungsten needles, even from valves from E12.5 embryos5. Because this additional step will lengthen the time of dissection and add significant technical difficulty, we suggest employing it as needed only. In our original study, we began with an immunohistochemical analysis that showed that Smad phosphorylation was only changed in the valve mesenchyme; thus, we were confident that any differences observed via western blot were due to changes in expression in the valve mesenchyme8. Further, because the developing outflow tract valves sit at the junction of the great arteries and the ventricles, performing a control blot for ventricular contamination using a ventricular myocardium-specific antibody such as ANP or MLC-2v is recommended.
Because the valves undergo dramatic reorganization during their development, we offer the following stage-specific recommendations for the cushions/valves and their surrounding tissue. For the early cushions (E9.5-11.5), both the outflow tract and atrioventricular cushions are easily visualized and dissected due to the translucency of the myocardium10; however, these embryos are small, and dissecting needles are recommended instead of forceps and microscissors. For mid-stage differentiation (E12.5-14.5)10, the semilunar valves (i.e., the aortic and pulmonary artery valves) will be more readily accessible than the atrioventricular valves (i.e., the mitral and tricuspid valves). However, the semilunar valves must be carefully dissected because the aorta and pulmonary artery are septating and rotating during this time frame11,12. Careful attention should be given to the rotation of the base of the aorta and pulmonary artery, and the midpoint between these arteries should be consistently identified. At later stages of valve maturation and condensation (E15.5 and later)10, the mitral and tricuspid valves can be teased away from inside the ventricles; however, the semilunar valves should be more easily accessible.
Western blot analysis is a long-standing technique for quantifying protein expression levels in adult cardiac valves13, where the biopsy size is large. In contrast, the complimentary early embryonic analyses have focused on either non-quantitative immunohistochemistry to detect protein expression or reverse transcription PCR, where the small starting amount of isolated mRNA can be amplified prior to analysis. These currently employed techniques provide some pertinent information regarding changes in signaling and gene expression but lack the ability to quantitatively assess protein levels. With this protocol, we can now add quantitative protein expression data to correlate with the quantitative mRNA expression data and immunohistochemical analyses.
This protocol compliments established techniques for isolating mRNA from the embryonic cardiac valves and imaging protein expression. As such, combining these techniques will allow for more complete analyses of the up- and down-regulation of signaling pathways, from mRNA to post-translational protein modification. Further, this technique allows for quantification of protein expression, which will enhance currently utilized immunohistochemistry techniques. Together, we believe that this technique will be of interest for any researcher interested in the cardiac valves or preparing other particularly small tissue explants for quantitative western blot analysis.

Being able to identify and quantify protein expression levels is a standard technique for animal- and cell-based experiments. However, despite a long-standing interest in early embryonic cardiac valve development, evaluating protein expression in this specific tissue during development is currently limited to immunohistochemistry in both the chick and mouse1,2. Part of the difficulty of quantifying protein expression in the developing valves of most model organisms (e.g., chick and mouse) is the small size of the valves, which limits the quantity of protein that can be obtained. Thus, for quantitative analyses, researchers typically rely on RNA extraction and amplification for subsequent quantitative PCR or microarray analysis2-5. However, RNA and protein expression levels are not wholly correlative6, so focusing on RNA expression cannot provide a rigorous account of the numerous changes that occur in any given signaling pathway at various times during developments. Based on this limit in the currently available methodology, the goal of this procedure was to develop a protocol for reliably obtaining sufficient amounts of protein from the developing embryonic mouse cardiac valve regions for quantitative analysis of changes that occur in various signaling pathways that are important in the maturation of this tissue.
Embryonic valves are already commonly dissected from mice for RNA isolation and subsequent gene expression analysis2-5. However, these studies have been limited to using gene expression as a read-out of signaling pathway activation, which does not allow the detection of detect post-translational protein modifications that may affect downstream signaling. Using the RNA isolation techniques as a starting point, we began with dissecting the regions of interest. Because our interest was detecting phosphorylated proteins that were indicative of signaling pathway activity during a specific period of aortic valve development (E13.5-14.5), we performed all dissections in phosphate-free Tris buffer and collected the valves in a Tris-based lysis buffer with phosphatase and protease inhibitors. In our specific case, only the aortic valve regions were collected, but the pulmonary valve region could easily be obtained at the same time. The valve regions were then homogenized and combined with a sample buffer that is currently used to study protein expression in adult cardiac valves7. By using small sample volumes (e.g., 2 &#956;l) and pooling valve regions from embryos with the same genotype, we were able to detect phosphorylated and nonphosphorylated proteins at embryonic day 13.5 8. Because the valve regions can be frozen and stored in lysis buffer, embryos can be genotyped if needed before pooling.
This technique broadens the set of tools that are available for evaluating cell signaling pathways during development and provides a quantitative compliment to immunohistochemistry, specifically for the developing cardiac valves. This technique should be of benefit not only to developmental cardiologists but also to all developmental biologists who work with early stage embryos are interested in regions that contain limited tissue.

<table><tbody><tr><td>Timed-pregnant mouse</td><td></td><td></td><td>To be dissected at the embryonic stage of interest</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>0.1 M Tris, pH 7.6</td><td></td><td></td><td></td></tr><tr><td>Microscissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine forceps, #5</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Dissecting needle holders</td><td>Ted Pella Inc.</td><td>13560</td><td></td></tr><tr><td>Dissecting needles</td><td>Ted Pella Inc.</td><td>13561-10</td><td></td></tr><tr><td>Micropipette, 20 &amp;mu;l, with tips</td><td></td><td></td><td></td></tr><tr><td>Lysis buffer</td><td></td><td></td><td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td>Add 1 tablet to 10 ml lysis buffer</td></tr><tr><td>TissueLyser LT</td><td>Qiagen</td><td>85600</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

Using this preparation technique, we were able to detect phosphorylated Smad1,5,8 (pSmad) in single aortic valve regions from E13.5 embryos. As shown in Figure 1A, the protein isolated from even a single valve region is sufficient to detect a faint pSmad band. Signal intensity increases proportionately to the number of valve regions that are pooled. Importantly, the pSmad/&#946;-actin ratio remains nearly constant across the different sample sizes (Figure 1C). Due to the low levels of protein available, the same blot cannot be stripped and reprobed for total Smad. However, a separate blot showing total Smad 1 and &#946;-actin shows the same expression pattern (Figure 1B).
These valve regions are almost entirely devoid of contaminating ventricular myocardium. Using this preparation technique in combination with samples of the right ventricle, we were unable to detect ventricular myocardium marker ANP in aortic valve regions, whereas this marker was readily detected in the ventricle (Figure 2). Further, the ventricular myocardial marker myosin light chain 2V is also readily detected in the ventricular sample but is barely detectable in the aortic valve regions. These results support the preciseness of the dissection.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51911/51911fig1highres.jpg" width="500" /> 
Figure 1.&#160;Smad phosphorylation in embryonic aortic valve regions.&#160;Samples were prepared with increasing numbers of pooled embryonic mouse aortic valve regions, ranging from 1 to 4 valve regions per sample. Each sample includes all proteins isolated from the entire valve region(s) in a total volume of 25-28 &#956;l. Prepared tissues were then subjected to western blot analysis for phosphorylated Smad1,5,8 (pSmad) (A), total Smad1 (C), and &#946;-actin (A, C). Protein was detected using Lumigen ECL Ultra and imaged with UVM VisionWorks software. The band intensity of pSmad and &#946;-actin is proportional to the amount of starting material, as quantified in (B) using ImageJ software. The data presented are the mean and standard deviation of two separate blots.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51911/51911fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51911/51911fig2highres.jpg" width="500" /> 
Figure 2.&#160;Aortic valve regions do not express ventricular myocardium markers.&#160;Samples were prepared with increasing numbers of pooled embryonic mouse aortic valve regions, ranging from 1 to 4 valve regions per sample, or with a sample of the right ventricle. Samples were processed as described in Figure 1 and blotted for the ventricular myocardium markers ANP (A) and myosin light chain (MLC)-2v (B). ANP is completely restricted to the ventricular sample, and MLC-2v is expressed at extremely low levels compared with the ventricular sample. &#946;-actin is shown as a loading control.

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

Nanyang Technological University

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9.4K Views.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Video: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Developmental Biology

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Medicine

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

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