We thank The Ohio State University Comprehensive Cancer Center Analytical Cytometry Core facility, specifically Katrina Moore, for technical assistance with FACs analysis. In addition we recognize Dr. William Pu and his group for their scientific insights. This work was supported by NIH HL091878 (JL) and The Heart Center at Nationwide Children&#8217;s Hospital.&#160;

1. Preparation of Equipment and Solutions
<ol>
	<li>Sterilize the dissection tools - fine tissue scissors to extract adult hearts and 2 fine forceps for dissection of the valvular region - by autoclaving in a covered instrument tray. Spray tools with 70% ethanol (EtOH) prior to dissection.</li>
	<li>Prepare all solutions immediately prior to the experiment and sterilize solutions by passing them through a sterile 0.2 &#956;m filter. Keep solutions on ice until use.
	<ol>
		<li>Sterile Dissociation Buffer (15 ml total/sample). Combine 1.2 ml collagenase IV, 300 &#956;l 2.5% trypsin and 150 &#956;l chick serum. Bring volume up to 15 ml with Hank&#8217;s Balanced Salt Solution (HBSS).</li>
		<li>Sterile Sorting Buffer (12 ml total). Combine 25 &#956;l 0.5 M ethylenediaminetetraacetic acid (EDTA) and 12 &#956;l DNase I (RNase-free). Bring volume up to 12 ml with HBSS.</li>
		<li>VEC Culture Media. Mix endothelial growth media according to the manufacturer&#8217;s instructions (see material spreadsheet) by adding the aliquoted components from the kit to 500 ml of EBM2 media.</li>
		<li>GFP Negative Culture Media (non-endothelial cell media). Mix 445 ml of Medium 199 1x with 5 ml of penicillin/streptomycin (pen/strep) (1% final concentration) and 50 ml FBS (10% final concentration).</li>
	</ol>
	</li>
</ol>
2. Dissection of the Valvular Region from Adult mice and E14.5 embryos
<ul>
	<li>All animal procedures were approved and performed in accordance with The Nationwide Children&#8217;s Hospital Research Institute IACUC guidelines.</li>
	<li>Tie2-GFP mice were purchased from Jackson Laboratories (stock number 003658)14 and were maintained as homozygous genotypes on an FVB/N background, and therefore ensuring that all off-spring express GFP in Tie2-positive endothelial cells.</li>
	<li>For FACS sorting, prepare one age matched negative sample that does not contain GFP such as C57/Bl6 to set GFP gate parameters for FACS sorting. NOTE: This protocol and representative results are based on using three, four-month-old adult mice or one litter at embryonic day (E) 14.5.</li>
</ul>
<ol>
	<li>Adult Mice: Immediately following sacrifice by CO2 anoxia, use dissection scissors to gently open the chest cavity and expose the heart and lungs. Once exposed, grip the heart with forceps at the great arteries and pull away from the body. Remove lung tissue if intact, and place hearts in cold HBSS to rinse. Remove the ventricle and pull away the atria leaving the atrioventricular canal &#8216;ring&#8217; and aortic regions intact. Make one incision down the side of atrioventricular canal to open up the &#8216;ring&#8217; and expose the valvular structures. Remove the myocardium and proximal aorta regions by gently teasing away with forceps. Identify the valve leaflets as white, dense tissue over the pink myocardium. Gently detach the chordae tendinae from the atrioventricular valves and remove as much of the remaining myocardium as possible. Be cautious to not pull or scrape the valve leaflets during this process since VECs may be dislodged. Place trimmed valvular regions in a 1.5 ml eppendorf tube containing 1 ml HBSS and keep on ice. NOTE: Careful dissection is critical to eliminate non-valvular endothelial cells that could contaminate the sample.</li>
	<li>Embryos: Sacrifice female mice 14.0 days after copulation plug is observed (morning of copulation plug = 0.5 days).
	<ol>
		<li>Immediately following sacrifice, dissect the uterus (containing embryos) and wash in cold HBSS. Dissect out individual embryos from the uterus and remove the embryonic yolk sac surrounding each embryo. Remove the hearts from each of the embryos and follow step 2.1. (NOTE: gently squeezing the embryo with forceps just below the upper appendages should help to expose the heart and make removal easier.)</li>
	</ol>
	</li>
</ol>
3. Cell Dissociation and Preparation for FACS
<ol>
	<li>Using sterile pipette tips, remove HBSS from the eppendorf tube containing the valvular regions.</li>
	<li>Replace with 1 ml dissociation buffer and 4 &#956;l DNase I.</li>
	<li>Rotate the tubes at 37 &#176;C for 7 min.</li>
	<li>Pipette up and down 3 times and then let the sample settle for 15 sec. Collect the supernatant (containing the dissociated cells) into a 15 ml conical tube.</li>
	<li>To stop the collagenase reaction, add 125 &#956;l of horse serum to the collection tube. Keep collection tube on ice.</li>
	<li>Repeat dissociation steps (3.2 - 3.5) nine times to collect nine fractions of supernatant.</li>
	<li>Pass the fractioned collection through a 70 &#956;m nylon filter into a new collection tube to ensure a single cell suspension by removing any debris and clumps.</li>
	<li>Spin the suspension at 400 g for 5 min to pellet the cells.</li>
	<li>Resuspend the cell pellet in 1 ml Sorting Buffer and keep on ice until FACS.</li>
</ol>
4. FACS Sorting GFP positive VECs
<ol>
	<li>Set gates for forward and side scatter to exclude debris and capture single cells; exclude doublets by using doublet discrimination gating. Record the negative control sample and define gate settings in order to compare and accurately define the GFP-positive cell population in the Tie2-GFP sample.</li>
	<li>Analyze GFP positive cells via FACS and refine gate settings.</li>
	<li>Sort the Tie2-GFP sample and collect both GFP-positive and GFP-negative cells. If cells will be used for RNA extraction, sort directly into the Sorting Buffer. To culture cells after FACS, collect GFP positive cells in a sterile tube containing 1 ml VEC media with 1 ml FBS, and collect GFP negative cells in 1 ml of non-endothelial media with 1 ml FBS. Use this 50% media/50% serum combination to improve cell viability. Keep on ice until post FACS analysis.</li>
</ol>
5. Post FACS Analysis
<ol>
	<li>RNA Extraction:
	<ol>
		<li>Immediately following FACS, centrifuge GFP positive and negative cells at 1,500 x g for 8 min.</li>
		<li>Carefully pipette the supernatant (sorting buffer) and discard.</li>
		<li>Resuspend the cell pellet (pellet is not visible for the sample sizes recommended here) in 200 &#956;l Trizol.</li>
		<li>Freeze at -80 &#176;C or begin standard phenol-chloroform extraction to isolate total mRNA as previously described by our lab15.</li>
		<li>Use 50-200 ng of RNA for cDNA synthesis using the Mastermix according to the manufacturer&#8217;s instructions.</li>
		<li>Subject cDNA to quantitative PCR amplification using IDT Primetime qPCR probes against mouse endothelial cell markers (CD31, von Willebrand Factor (vWF)), valve interstitial cell markers (&#945;-smooth muscle actin (&#945;-SMA), Periostin (Postn)), myocyte markers (Mhc6, Mhc7) and GAPDH.</li>
	</ol>
	</li>
	<li>Culturing Murine VEC:
	<ol>
		<li>After FACS sorting and cell collection (step 4.3), centrifuge cells at 300 x g for 5 min.</li>
		<li>Carefully pipette off the supernatant (sorting buffer + media) and discard.</li>
		<li>Resuspend the GFP-positive cell pellet in 1 ml of VEC media and the GFP-negative pellet in 1 ml non-endothelial media.</li>
		<li>Plate each sample in one well of a plastic chamber slide and grow until confluent. Culture for about 1 week for GFP negative cells to become confluent and more than 2 weeks for GFP positive cells to become confluent (from a sample of 3, adult mice). Change media every two days.</li>
	</ol>
	</li>
	<li>Immunofluorescent Staining:
	<ol>
		<li>Remove media from cells and fix in 4% paraformaldehyde (PFA) for 30 min at RT. Wash fixed cells three times for 5 min each in Phosphate Buffered Saline solution (PBS).</li>
		<li>Remove chamber wells from slide and block in 5% bovine serum albumin/1x PBS for 1 hr at RT.</li>
		<li>Incubate slides O/N at 4 &#176;C with primary antibodies (CD31, 1:1,000 or &#945;-smooth muscle actin (&#945;-SMA), 1:100). Wash 3x for 5 min in PBS.</li>
		<li>Dilute the secondary antibody (Alexa-Fluor 568 Goat anti-Rat) 1:400 in PBS, add to slides, and incubate 1 hr at RT. Rinse slides 3x for 5 min in PBS.</li>
		<li>Mount in Vectashield-containing DAPI and incubate 1 hr at 4&#176; C prior to viewing.</li>
	</ol>
	</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+deletion+of+murine+Jag1+leads+to+valve+calcification+and+congenital+heart+defects+associated+with+Alagille+syndrome.">Endothelial deletion of murine Jag1 leads to valve calcification and congenital heart defects associated with Alagille syndrome.</a> Development. 139, 4449-4460 (2012).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arteriosclerosis, thrombosis, and vascular biology. 31, 598-607 (2011).</li><li>Gould, R. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+valvular+endothelial+cells.">Isolation of valvular endothelial cells.</a> Journal of Visualized Experiments : JoVE. , (2010).</li><li>Butcher, J. T., Penrod, A. M., Garcia, A. J., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arteriosclerosis, thrombosis, and vascular biology. 24, 1429-1434 (2004).</li><li>Cheung, W. Y., Young, E. W., Simmons, C. 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The authors have nothing to disclose.

Here we describe for the first time, a novel method for the isolation of embryonic and adult murine VECs from Tie2-GFP mice. While this mouse line has been extensively used for the isolation of endothelial cell populations, this is the first report showing selective isolation of VECs. Due to the fragility of the VEC population, we have developed a stringent protocol that allows for single cell isolation of GFP positive (and GFP negative) cells from heart valves of embryonic and adult mice. Compared to the original publication using whole embryos or organs from Tie2-GFP mice14, we have optimized collagenase and dissection steps based on the low abundance of this fragile endothelial cell population to isolate a select population of cells.
VEC isolations have previously only been reported in large animal models with limited genetic and biomolecular tools. These tools are well established in mice and therefore, the ability to isolate murine VECs allows for an expanded set of experimental designs to be used for heart valve research. Therefore, a significant advantage of this approach is that VECs can be isolated temporally from wild type mice, and models of valve disease and injury. A second advantage is that VECs can be isolated and analyzed almost immediately after dissection, preserving expression patterns that reflect the in vivo situation more accurately. Further, this isolation protocol is sufficient to eliminate cell culture for number expansion and therefore potential phenotypic changes induced by the in vitro environment are prevented.
Despite the novelties and experimental benefits of this protocol, we recognize that limitations still exist. First, the size of the VEC population within murine valves is very small and therefore, multiple timed embryonic litters are needed in order to generate sufficient RNA for gene expression analysis. While this can be overcome using multiple breeders, it could have an impact on the application of some post-isolation analysis tools. This limitation has been a challenge particularly for establishing confluent cultures of GFP-positive VECs in order to perform more thorough analyses of VEC phenotypes including molecular profiles and functional assays. Therefore we acknowledge that not all difficulties have been overcome by our approach but this is an area of interest that we are striving to overcome.
This approach of isolating VECs from valvular regions introduces the possibility of contamination from Tie-GFP-positive, non-valvular endothelial cells of the endocardium, or vascular structures within the ventricular myocardium. To date, molecular distinction of VECs from other cardiac endothelial cell populations have not been identified. However an enhancer region of the Nfatc1 gene has been identified and shown to specifically label VECs that do not undergo EMT, and no other endothelial cell population within the heart18. As a Cre model (Nfatc1enCre) is available, future studies could utilize the specificity of this line to minimize contamination risks. In addition to non-valvular Tie2-GFP-positive cells, there is always the possibility of contamination from myocytes at the point where the valve leaflets attach to the annular region of the septal and mural myocardial walls. In data not shown here, we initially began studies to isolate VECs from dissected valvular regions using antibody-conjugated beads coated with anti-GFP and anti-CD31. While this approach was successful for isolating the endothelial cells, we experienced significant cell clumping from adjacent, non-endothelial cell types and therefore VICs and myocardial cells contaminated our experimental sample. This has been avoided using FACS analysis as parameters have been set to isolate only single cell suspensions and PCR analysis to detect expression of myocyte-specific genes has controlled for this limitation (Figure 3). While our contamination can be deemed as minimal, it remains a potential experimental complexity which could be avoided in the future with the double selection of GFP and endothelial cell-specific surface markers.
Using this protocol, we have successfully isolated VECs from embryonic and adult mice and provided examples of how this approach can be used for RNA isolation and cell culture of GFP positive and GFP negative cell populations. However, this approach is not limited to these applications and can be used for a plethora of molecular, cellular and functional approaches. In addition, the Tie2-GFP background can be bred with genetic mouse models that will allow for comparative studies of VEC populations in health and disease. The development of this novel methodology will, for the first time, allow for focused studies examining the contribution of VECs in valve development and maintenance and could reveal previously unappreciated mechanisms of endothelial-dependent valve disease.

The mature valve is composed of three stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and encapsulated by a single layer of heart valve endothelial cells (VECs)1. The role of the ECM is to provide all the necessary biomechanical properties to withstand constant changes in hemodynamic force during the cardiac cycle. Turnover of the valve ECM in the adult valve is tightly regulated by VICs that are largely quiescent and fibroblast-like in the absence of disease. In addition to the VIC population, heart valve endothelial cells (VECs) form an uninterrupted endothelium over the surface of the valve cusps2. While the importance of ECM and VICs for valve structure and function have been described by us and others, the role of VECs is less well known. However, this comparatively small cell population is critical for valve formation in the embryo and is described as dysfunctional in valve disease.
Heart valve formation in the embryo begins when a subset of endothelial cells within the atrioventricular canal and outflow tract regions undergo endothelial to mesenchymal transformation (EMT) and form swellings known as endocardial cushions1,3. The newly transformed mesenchymal cells within these structures later differentiate into VICs and form the mature valves. Once EMT is complete, endothelial cells surrounding the cushions, termed VECs, form an uninterrupted endothelial cell layer that protects the mature valve against injury. In addition, VECs sense the hemodynamic environment and have been shown to molecularly communicate with underlying VICs to regulate ECM homeostasis1,3. In diseased valves, the VEC monolayer is disrupted in association with abnormal changes in ECM organization and altered biomechanics4,5. In addition, studies in mouse models suggest that VEC dysfunction is the underlying cause of valve disease1,6-9. As VECs play an important role in valve development, maintenance, and disease, it is important that we fully define their temporal phenotypes in order to advance the field and understand mechanisms of disease.
Previous work by several labs has successfully isolated VECs from porcine and ovine models10-12. Due to the large size of these valves, isolations through swabbing and/or enzymatic digestion, followed by a number of different isolation methods including magnetic bead cell separation and single cell clonal expansion has been effective to generate pure populations11-13. However, these models can be restrictive due to the incomplete annotation of the pig and sheep genomes limiting the availability of molecular tools in addition to the high costs. Therefore experimentation of porcine and ovine VECs after isolation can be restrictive. Mouse models are preferable due to the many possibilities for genetic manipulation and molecular tools in the embryo and adult, but to date, no VEC isolations in small animal models have been reported. This is likely due to the difficulty of working with small tissue samples that include a minority cell population that currently lack unique identity of VEC-specific markers, thereby preventing antibody-based isolation methods.
In this article, we report a new method for direct isolation of murine VECs at embryonic and adult stages. This protocol takes advantage of Tie2-GFP mice, which express GFP in all endothelial cell types and have been extensively used to study endothelial cell populations14. However, the novelty of this current study is that these mice, for the first time have been utilized to isolate endothelial cells from the valves. By careful dissection of the valve tissue and a series of nine enzymatic digestions followed by FACS sorting, VECs can be isolated and used for various experimental techniques including RNA extraction and culture, directly following sorting.

<table><tbody><tr><td>Alexa Fluor 568 Goat Anti-Rat IgG (H+L)</td><td>Life Technologies</td><td>A-11077</td><td></td></tr><tr><td>70 &amp;mu;m nylon cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>2.5% Trypsin</td><td>Invitrogen LT</td><td>15090-046</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V, Heat Shock Treated</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>CD31 rat anti-mouse antibody</td><td>BD Pharmingen</td><td>553370</td><td></td></tr><tr><td>Chick Serum</td><td>Invitrogen LT</td><td>16110-082</td><td></td></tr><tr><td>Collagenase IV</td><td>Invitrogen LT</td><td>17104-019</td><td>Prepared according to manufactuer&#039;s instructions</td></tr><tr><td>DNase I, RNase free (10,000 Units)</td><td>Roche</td><td>4716728001</td><td></td></tr><tr><td>EBM-2</td><td>Lonza</td><td>CC3156</td><td>For VEC media</td></tr><tr><td>EDTA, 0.5 M Solution&amp;nbsp;</td><td>Hoefer</td><td>03-500-506</td><td></td></tr><tr><td>EGM2 SingleQuot, Bulletkit</td><td>Lonza</td><td>CC-4176</td><td>For VEC media</td></tr><tr><td>Fetal Bovine Serum (FBS), Heat Inactivated</td><td>Hyclone</td><td>SH3007103IH</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>Horse Serum</td><td>Invitrogen LT</td><td>16050-130</td><td></td></tr><tr><td>Hybridization Oven</td><td>VWR</td><td>230301V</td><td></td></tr><tr><td>Medium 199 1X</td><td>Corning cellgro</td><td>10-060-CV</td><td></td></tr><tr><td>Paraformaldehyde 16% Solution EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Pen/Strep</td><td>MP-Biomed</td><td>ICN1670049</td><td></td></tr><tr><td>Permanox sterile chamber slide with cover</td><td>Thermo-Scientific</td><td>177429</td><td></td></tr><tr><td>Phosphate-Buffered Saline, 1X</td><td>Corning cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>PrimeTime Mini qPCR Assay</td><td>Integrated DNA Technologies</td><td>Acta2 (&amp;alpha;SMA), GAPDH, Myh6, Myh7, POSTN, CD31, vWF</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Applied Biosystems</td><td>4376600</td><td></td></tr><tr><td>SuperScript VILO Mastermix</td><td>Invitrogen LT</td><td>11755050</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Tie2-GFP mice</td><td>Jackson Laboratories</td><td>3658</td><td></td></tr><tr><td>Tissue forceps #5 11 cm</td><td>Dumont</td><td>14096</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>15596018</td><td></td></tr><tr><td>&amp;alpha;-SMA anti-mouse antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2547</td><td></td></tr><tr><td>VECTASHIELD HardSet Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr></tbody></table>

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.

Tie2-GFP cells co-localize with endothelial cell markers in the embryonic and adult heart valves.
In order to confirm the specificity of Tie2-GFP expression in VECs from embryonic and adult mice, immunofluorescence was performed to determine co-localization with the endothelial cell marker, CD31 in tissue sections prepared from E14.5 and 3 month old adult Tie2-GFP mice using methods previously published by our lab16. As shown in Figure 1, VECs co-express GFP (Figure 1 A,B,E,F) and CD31 (Figure 1 C,D,E,F) at both adult (Figure 1 B,D,F) and embryonic (Figure 1 A,C,E) stages, therefore validating our model for subsequent VEC isolation.
FACS analysis identified distinct GFP-positive and GFP-negative cell populations in embryonic and adult heart valves isolated from Tie2-GFP mice.
Wild type C57/Bl6 mice were used to set GFP parameters and approximately 2% of single-gated cells from Tie2-GFP mice show a significant enrichment in GFP-positive cells by FACS analysis in both embryonic and adult samples (Figure 2). Based on co-localization studies shown in Figure 1, these cells are considered VECs and yield an average of 61,800 total cells (samples range from 39,000-77,000 cells/sample) in adult samples (n = 3), and 8,928 cells (8,015-11,000 cells/litter) from one litter of E14.5 embryos.
PCR analysis confirms enrichment of endothelial cell markers in GFP-positive cells and valve interstitial cell markers in GFP-negative cells isolated from Tie2-GFP mice.
Gene expression analysis of GFP positive and negative cell populations by qPCR following FACS show distinct molecular profiles. Compared to GFP negative cell populations isolated from Tie2-GFP embryos and adults, GFP positive cells are enriched for expression of endothelial cell markers CD31 and vWF, (Figure 3). Further, expression of myocyte markers (Myh6, Myh7) in these cell populations is very low, demonstrating minimal contamination of non-endothelial cells. In contrast, GFP negative cells isolated from adult Tie2-GFP mice and E14.5 embryos are enriched for valve interstitial cell (VIC) markers, &#945;-sma and Periostin (POSTN) relative to GFP positive cells. Enrichment of these markers is higher in GFP negative cells isolated from E14.5 samples compared to adults due to the quiescent phenotype of adult VICs and therefore low expression of activated markers.
GFP-positive cells isolated from Tie2-GFP adult mice can be cultured in vitro.
The ability to culture GFP positive and GFP negative cells isolated from adult samples was examined based on cell morphology and immunohistochemical staining. Using protocols previously described by us17 we show in Figure 4 that GFP positive cells appear round in morphology (Figure 4A) and co-express GFP with the endothelial cell marker CD31 after two weeks in culture (Figure 4C). In contrast, non-endothelial cells are negative for GFP, display a mesenchymal-like morphology (Figure 4B) and express the VIC marker &#945;-SMA after nine days (Figure 4D).
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51860/51860fig1highres.jpg" width="400" /> 
Figure 1. Tie2-GFP-positive cells co-localize with CD31 in embryonic and adult heart valves. Immunofluorescence to show association of (Tie2-)GFP expression (A, B) with the endothelial cell marker, CD31 (C, D) in the septal leaflets of the mitral valve at E14.5 (A, C, E) and adult (B, D, F) stages. The valve region is highlighted in A, C, E. (E, F) Merged images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig1highres.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/51860/51860fig2highres.jpg" width="500" /> 
Figure 2. Tie2-GFP-positive VECs can be identified by FACS. Compared to age-matched C57/Bl6 controls (A, C), valves isolated from Tie2-GFP embryos (B) and adults (D) contain a distinct GFP-positive cell population, as indicated in green. GFP-negative events, shown in red were collected as a control. Numbers in (B) and (D) indicate the average % of GFP-positive cells from the total number of events sorted by FACS (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51860/51860fig3highres.jpg" width="500" /> 
Figure 3. GFP-positive cells are enriched for endothelial cell markers whereas GFP-negative cells express genes associated with valve interstitial cells. Representative qPCR of endothelial cell markers (CD31, von Willebrand Factor (vWF)) and adult (Myh6) and fetal (Myh7) myocyte markers in GFP-positive cells isolated from valvular regions of E14.5 (A) and adult (B) Tie2-GFP mice. Note enrichment of endothelial cell markers and very low levels of myocyte-associated genes. (C, D) Fold changes in expression of valve interstitial cell markers &#945;-sma and Postn in isolated GFP-negative cells from E14.5 (C) and adult (D) Tie2-GFP mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51860/51860fig4highres.jpg" width="500" /> 
Figure 4. Tie2-GFP-positive cells maintain expression of endothelial cell markers in vitro. Following FACS, GFP positive (A, C) and negative (B, D) cell populations from adult mice were cultured until confluent and subject to phase contrast imaging (A, B) and fluorescent immunostaining (C, D). GFP-positive cells express CD31 (red) (C) after 10 days of culture. In contrast, GFP expression was not detected in the negative cell population, which stained positive for &#945;-SMA, a marker of activated VICs (D).

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Isolation of Murine Valve Endothelial Cells

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-of-murine-valve-endothelial-cells

Nanyang Technological University

Research

JoVE Journal

Biology

14.2K Views.  The Ohio State University. 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.

Video: Isolation of Murine Valve Endothelial Cells

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, ...

Developmental Biology

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

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) ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Isolation of Murine Valve Endothelial Cells

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