This work was supported by NIH grants R01HL108735, R01HL145170, R01HL106089 (to Z.B.C.); DP1DK126138 and DP1HD087990 (to S.Z.); an Ella Fitzgerald Foundation grant and a Wanek Family Project (to Z.B.C.); and a Human Cell Atlas seed network grant (to Z.B.C. and S.Z.). Research reported in this publication included work performed in the Integrative Genomics Core at City of Hope supported by the National Cancer Institute of the National Institutes of Health under award number P30CA033572. The authors would like to thank Dr. Ismail Al-Abdullah and Dr. Meirigeng Qi of the islet transplantation team at City of Hope for isolation of human tissues, Dr. Dongqiang Yuan at City of Hope for his assistance with scRNA-seq analysis, and Dr. Marc Halushka at the Division of Cardiovascular Pathology, Johns Hopkins University School of Medicine for his invaluable insights into vascular histology.

Human tissue studies were conducted on deidentified specimens obtained from the Southern California Islet Cell Resource Center at City of Hope. The research consents for the use of postmortem human tissues were obtained from the donors&#39; next of kin, and ethical approval for this study was granted by the Institutional Review Board of City of Hope (IRB No. 01046).
1. Physical dissociation (estimated time: 1-2 h)
<ol>
	<li>Place a fresh artery on a 10 cm dish and wash with sterile Dulbecco&#39;s phosphate-buffered saline (D-PBS).</li>
	<li>With sterile forceps and dissection scissors, remove the fat and outer connective tissues until the vessel is clean. Measure the length of the vessel with a ruler placed outside of the dish and take pictures for records (Figure 1A).</li>
	<li>Pre-warm appropriate digestion buffer to 37 &#176;C: for scRNA-seq use cell-dissociation enzyme; for cell culture assays use 1-6 mg/mL of Collagenase D, 3 mg/mL of bacteria-derived protease, 100 mM of HEPES, 120 mM of NaCl, 50 mM of KCl, 5 mM of glucose, with 1 mM of CaCl26,38 (pH 7.0, does not require adjustment) added 5 min prior to use.</li>
	<li>Take the mesenteric artery (typically with a diameter of 6-8 mm) and cut lengthwise with scissors to open the vessel lumen vertically.</li>
	<li>Using needles, attach the vessel onto black wax on all four corners, leaving the intima exposed (Figure 1B).</li>
	<li>Add 1 mL of pre-warmed digestion buffer to intima. Take a sterile scalpel and scrape the lumen of the vessel gently twice. 
	NOTE: The purpose of this procedure is to dissociate the intimal layer, which may need practice. Enough force needs to be exerted to remove endothelium, but not so much that the deeper layers of tissue are also removed, which will reduce the EC representation.</li>
	<li>Transfer the digestion buffer into a 5 mL tube. Add 1 mL of digestion buffer to the intima and pipette up and down carefully to collect the remaining cells and add to the 5 mL tube. Incubate cells at 37 &#176;C, rotating at 150 rpm for 5 min.</li>
	<li>Add 2 mL of M199 medium (or D-PBS if proceeding with scRNA-seq) to the cell suspension to quench the enzymatic reaction. Mix gently and centrifuge at 4 &#176;C, 600 x g for 5 min.</li>
	<li>Remove the supernatant and store the supernatant separately to culture as a control to observe if all cells are captured in the pellet in step 1.8. Resuspend the cell pellet in 1 mL of M199 medium (or D-PBS if proceeding with scRNA-seq).</li>
	<li>Assess the cell viability by mixing 10 &#181;L of trypan blue with 10 &#181;L of cell stock. Observe the cell morphology and count the cells using a hemocytometer. 
	&#8203;NOTE: At this point, cells can be used for protein or RNA quantification or cultured in vitro using EC culture media following standard protocols39. Alternatively, they can be prepared for sequencing as described in the next section.</li>
	<li>For the culture, coat two wells of a 6-well plate by pipetting 500 &#181;L of attachment reagent into wells for 30 min at room temperature. After removal of attachment reagent and washing with sterile D-PBS, dispense the full cell stock into one well, and the supernatant kept as a control into the second well.</li>
</ol>
2. Preparation for scRNA-seq studies (estimated time: 3-4 h)
<ol>
	<li>Centrifuge the cell stock from step 1.11 at 4 &#176;C, 600 x g for 5 min. Remove the supernatant and resuspend the pellet gently with a P1000 pipette and wide-bore tip in 1 mL of 0.04% bovine serum albumin (BSA) in D-PBS. Mix well to ensure a single-cell suspension.</li>
	<li>Pass the solution through a 40 &#181;m strainer to remove cell debris. If debris remains, pass through a second strainer into 4 mL of 0.04% BSA in D-PBS.</li>
	<li>Centrifuge at 4 &#176;C, 600 x g for 5 min. Remove the supernatant and resuspend the pellet in 500 &#181;L of 0.04% BSA in D-PBS using a P1000 pipette with a wide-bore pipette tip. Mix well to ensure a single-cell suspension.</li>
	<li>Assess the cell viability by mixing 10 &#181;L of cell stock with 10 &#181;L of trypan blue. Using a hemocytometer, observe the morphology, determine whether there is a single-cell suspension without clusters, check for tissue debris, and calculate the number of living and dead cells.</li>
	<li>If cells are clustered or debris remains, repeat washing with 0.04% BSA in D-PBS or pass through 40 &#181;m strainer again. 
	NOTE: While this will reduce the yield, it is important that cells exist in a clean single-cell suspension for optimal sequencing.</li>
	<li>The single-cell suspension can be used for scRNA-seq.</li>
</ol>
3. Spatial transcriptome profiling (estimated time: 3-4 h)
<ol>
	<li>Add isopentane (2-methylbutane) to a metal canister and chill in liquid nitrogen (LN2) or dry ice (LN2 is preferred as it will bring the temperature lower than dry ice). Pour optimal cutting temperature compound (OCT) in wells of labeled plastic cryomolds taking care to not create bubbles and remove those that appear. Leave cryomold on dry ice to chill.</li>
	<li>Cut at least two coronal sections, 1 cm in length of the vessel. Using long (12&#34;) forceps, submerge the tissue in isopentane until frozen. Quickly submerge the tissue in OCT at orientation with the lumen visible in the center. Take care to remove any bubbles, especially those next to the tissue.</li>
	<li>Using long (12&#34;) forceps, grasp the cryomolds, and hold them in the metal canister. While the base of the molds should be in the liquid, it should not be too deep to allow isopentane to run over the tissue. Observe the freezing, the OCT will become white progressively from the outside in 1-2 min.</li>
	<li>Store these sections in a sealed container at -80 &#176;C for up to 6 months. 
	NOTE: Maintain samples on dry ice at all times and do not allow more than one freeze-thaw cycle. This tissue can be used for histological analysis, RNA/DNA fluorescence in situ hybridization (FISH), or spatial transcriptomics, which will be described now.</li>
	<li>Set up the cryostat temperature to -20 &#176;C for the chamber and -10 &#176;C for the specimen head. Equilibrate OCT embedded vessel sections, knives, brushes, and slides to -20 &#176;C in the cryostat for approximately 30 min. While equilibrating, clean the machine and all equipment that may touch the sections, including the blade, with 70% ethanol followed by RNase decontamination solution.</li>
	<li>Attach the sample by dispensing a small amount of OCT onto the circular cryostat block and placing the sample on top before it freezes. Place the block into the center of the specimen head and screw the block in place using a tall black handle on the left. Cut away any excess OCT surrounding the vessel.</li>
	<li>Setting the cutting thickness to 10 &#181;m on the cryostat, cut approximately 60 sections and place them in a pre-cooled 1.5 mL tube. These sections will be used to assess the RNA quality. Upon removal from cryostat, immediately add 1 mL of RNA extraction reagent and vortex until the sections are completely dissolved.</li>
	<li>Add 200 &#181;L of chloroform and mix until the solution resembles a strawberry milkshake. Centrifuge at 11,200 x g for 10 min at 4 &#176;C.</li>
	<li>Collect the aqueous phase (clear phase on top of white and pink layers) and add 500 &#181;L of isopropanol to it. Mix by inverting the tubes over 10 times and place at -80 &#176;C for at least 20 min.</li>
	<li>Centrifuge at 11,200 x g for 10 min at 4 &#176;C. Dissolve the purified RNA pellet in 5 &#181;L of RNase-free water. Examine the RNA Integrity Number (RIN). 
	NOTE: Samples with RIN &#8805; 7 are preferred, but RIN &#8805; 6 is acceptable. The number of tissue sections and volume of solution for RNA dissolving may require optimization depending on the system used to ensure the final RNA concentration is within the RIN function range to allow for accurate RIN evaluation.</li>
	<li>Practice cutting and placing sections properly within the fiducial frames using plain glass slides before proceeding to commercially available tissue optimization or gene expression slides. This can be achieved by drawing 6.5 mm x 6.5 mm squares on a plain glass slide, cooling to -20 &#176;C in the cryostat, and placing sections in this capture area.</li>
	<li>Cut 10 &#181;m sections with anti-roll plate in place, flip and carefully flatten by gently touching the section through the surrounding OCT.</li>
	<li>Using RNase-free cryostat brushes place the tissue section within the square, using only the surrounding OCT. Immediately place one finger (in gloves) on the backside of the capture area to melt the section to the slide.</li>
	<li>Once the section has adhered, place the slide onto the cryobar to allow the section to freeze. Care must be taken to avoid tissue folding and tissue overlying the demarcated edges of the fiducial frame. If necessary, cut the tissue into halves or quarters along the lumen using a blade to ensure the vessel section fits within the 6.5 mm x 6.5 mm frame.</li>
	<li>Cut 10 &#181;m sections of tissue as per 3.12-3.14 onto tissue optimization or gene expression slides. Transfer the slide to a slide mailer placed on dry ice. Store slides at -80 &#176;C for up to 4 weeks before proceeding with published spatial protocols33,34.</li>
</ol>
4. Sequencing data analysis (estimated time: up to 1 week depending on familiarity with software)
NOTE: For spatial transcriptomic analysis only, skip to step 4.8. scRNA-seq data is processed using the standardized pipeline aligned to human hg38 reference transcriptome. The R package Seurat (v3.2.2) is used to analyze scRNA-seq data following published guidelines40.
<ol>
	<li>Filter using well-established quality control metrics: rare cells with very high numbers of genes (potentially multiplets) and cells with high mitochondrial percentages (low-quality or dying cells often present mitochondrial contamination) are removed.</li>
	<li>Normalize data using &#34;sctransform&#34;, a method to improve sample integration compared to log-normalization. These normalized data are used for dimensionality reduction and clustering, while log-normalized expression levels are used for analysis based on gene expression levels, such as cell-type classification41.</li>
	<li>Select marker genes per cell type, for example, platelet endothelial cell adhesion molecule-1 (PECAM1) and von Willebrand factor (VWF) for ECs, lumican (LUM) and procollagen C-Endopeptidase Enhancer (PCOLCE) for fibroblasts, B-cell translocation gene 1 (BTG1) and CD52 for macrophages, C1QA and C1QB for monocytes, and myosin heavy chain 11 (MYH11) and transgelin (TAGLN) for vascular smooth muscle cells36.</li>
	<li>Compute the average expression level across single cells between each pair of markers in order to have a single marker with an average expression level associated with each cell type42.</li>
	<li>Apply a Gaussian Mixture Model (GMM) with two components to the expression data of each marker across single cells in order to separate cells into two sets, namely, highly expressing the marker and lowly expressing the marker. In this way, for each marker, each single cell is assigned to one of the two components42.</li>
	<li>Use statistical enrichment for the set of marker genes, a Fisher&#39;s exact test, to assign a cell type to each cluster. By doing so, a set of p-values are obtained per cluster, each corresponding to a cell type. The cell type with the lowest p-value is assigned to that cluster.</li>
	<li>Process the spatial transcriptomic data using the Space Ranger pipelines resulting in spatial de-barcoding and generation of quality control (QC) metrics, including total aligned reads to hg38 human transcriptome, the median number of Unique Molecular Identifier (UMI) or genes per spot35. 
	NOTE: The R package Seurat (v3.2.2) is used to analyze the Space Ranger-processed data following published guidelines40.</li>
	<li>Normalize data using &#34;sctransform&#34;, a method which is demonstrated to improve the downstream analysis compared to log-normalization given the heterogeneity of the tissue and the very high variance in counts across the spots.</li>
</ol>

S.Z. is a founder and board member of Genemo, Inc.

The presented workflow details a set of techniques to profile ECs from a single piece of human artery with single-cell and spatial resolution. There are several critical steps and limiting factors in the protocol. One key to transcriptome profiling is the freshness of the tissue and RNA integrity. It is important to maintain tissues on ice as much as possible prior to processing to minimize RNA degradation. Typically, the post-mortem tissues are processed between 8-14 h after the time of death. However, beginning the iso...

Lining the lumen of blood vessels, endothelial cells (ECs) are crucial regulators of vascular tone and tissue perfusion. ECs are remarkable in their ability to react to the extracellular environment and adapt to changes in the dynamics and composition of blood flow. These dynamic responses are mediated through a network of intracellular signaling events, including transcriptional and post-transcriptional modulations with spatio-temporal resolution. The dysregulation of these responses is implicated in many pathologies, including but not limited to cardiovascular disease, diabetes, and cancer1,2.
A large proportion of studies make use of cell lines or animal models to interrogate EC transcriptome. The former is a useful tool, given the relative ease of use and inexpensiveness. However, serial culturing can introduce phenotypic alterations to ECs, such as fibroblastic features and a lack of polarization, disconnecting them from their in vivo state3. The primary cells, e.g., human umbilical vein EC (HUVEC) have been a popular choice since the 1980s but are derived from a developmental vascular bed that does not exist in adults, thus unlikely to fully represent mature ECs. Animals, especially mouse models, better represent the physiological or pathophysiological environment of ECs and allow interrogation of transcriptomes as a result of genetic perturbation. Murine ECs can be isolated from various tissues, including the aorta, lungs, and adipose tissues using enzyme-based procedures4,5,6,7. However, the isolated cells cannot be used for multiple passages unless transformed6 and are often limited in numbers, which requires pooling from multiple animals5,8,9.
The advent of new technologies exploring the vessel architecture at a transcriptomic level, particularly with single-cell resolution, has enabled a new era of endothelial biology by revealing novel functions and properties of ECs5, 10,11,12,13,14. A rich resource built by Tabula Muris investigators collected single-cell transcriptomic profiles of 100,000 cells including ECs across 20 different murine organs15, which revealed both common EC marker genes and unique transcriptomic signatures with inter-and intra-tissue differences5,13. Nevertheless, there are clear differences between mouse and human in genome, epigenome, and transcriptome, especially in the non-coding regions16,17,18. These aforementioned drawbacks emphasize the importance of analysis of ECs using human samples in order to gain a faithful profile of ECs in their native state in health and disease.
Most EC isolation methods rely on physical dissociation through homogenization, finely cutting, and mincing the tissue before incubation with proteolytic enzymes for differing times. The enzymes and conditions also vary considerably between tissue types, from trypsin to collagenase, used alone or in combination19,20,21. Further antibody-based enrichment or purification are often included to increase the purity of ECs. Typically, antibodies against EC membrane markers, e.g., CD144 and CD31 are conjugated to magnetic beads and added to the cell suspension22,23. Such a strategy can be generally adapted for EC isolation from multiple human and mouse tissues, including the techniques introduced in this protocol.
In their native state, ECs interact with multiple cell types and may exist in vascular niches where cell proximity is crucial for function. While single-cell and single-nuclear RNA-sequencing (scRNA and snRNA-seq) studies have been paramount to the recent breakthroughs in describing EC heterogeneity, the dissociation process disrupts tissue context and cell-cell contact, which are also important to understand EC biology. Developed in 2012 and named Method of the Year in 202024, spatial transcriptome profiling has been utilized to profile global gene expression while retaining the spatial features in various tissues including brain25, tumor26, and adipose tissue27. The technologies can be targeted, using specialized probes specific for particular RNA sequences attached to affinity reagents or fluorescent tags, thus detecting select genes at subcellular resolution28,29,30,31. They can also be untargeted32,33, typically using spatially barcoded oligonucleotides to capture RNA, which together get converted to cDNA for subsequent seq library preparation and hence has the advantage of deducing whole tissue gene expression in an unbiased manner. However, spatial resolution is not currently achieved at a single cellular level with commercially available technologies. This can be overcome to some extent with data integration with scRNA-seq data, ultimately allowing the mapping of single-cell transcriptome in a complex tissue context while retaining its original spatial information34.
Herein, a workflow is described to profile EC transcriptome using human superior mesenteric artery, a peripheral artery that has been used to study vasodilation, vascular remodeling, oxidative stress, and inflammation35,36,37. Two techniques are described: 1) to isolate and enrich ECs from the intima of blood vessels combining mechanical dissociation and enzymatic digestion suitable for single-cell transcriptome sequencing or subsequent in vitro culture; 2) to prepare arterial sections for spatial transcriptome profiling (Figure 1). These two techniques can be performed independently or complementarily to profile ECs and their surrounding cells. Furthermore, this workflow can be adapted for use on any medium or large artery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL micro-centrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>23G needles</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Thermo Fisher</td>
			<td>AC327270010</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m strainer</td>
			<td>Fisher</td>
			<td>14100150</td>
			<td></td>
		</tr>
		<tr>
			<td>4200 TapeStation System</td>
			<td>Agilent Technologies</td>
			<td>G2991BA</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tube</td>
			<td>Thermo Fisher</td>
			<td>14282300</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner Bio-One</td>
			<td>07-000-208</td>
			<td></td>
		</tr>
		<tr>
			<td>Attachment factor</td>
			<td>Cell Applications</td>
			<td>123-500</td>
			<td>Attachment reagent in the protocol</td>
		</tr>
		<tr>
			<td>Black wax</td>
			<td></td>
			<td></td>
			<td>Any commercial black wax can be used</td>
		</tr>
		<tr>
			<td>Bovine serum albumin heat shock treated</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>BP510</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>C607</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat brushes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>MT25950CQC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>Bacteria-derived protease in the protocol</td>
		</tr>
		<tr>
			<td>Disposable Safety Scalpels</td>
			<td>Myco Instrumentation</td>
			<td>6008TR-10</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Thermo Fisher</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity D1000 sample buffer</td>
			<td>Agilent Technologies</td>
			<td>5067-5603</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity D1000 screen tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>Kept at 37 &#176;C 5% CO2</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>BP26324</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma Aldrich</td>
			<td>M2520-10X</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal cannister</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>To assess cell morphology</td>
		</tr>
		<tr>
			<td>Microvascular endothelial culture medium</td>
			<td>Cell Applications</td>
			<td>111-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 44/44R Orbital shaker</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature compound</td>
			<td>Fisher</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cryomolds</td>
			<td>Fisher</td>
			<td>22363553</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA screen tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA screen Tape sample buffer</td>
			<td>Agilent Technologies</td>
			<td>5067-5577</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase ZAP</td>
			<td>Thermo Fisher</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Takara</td>
			<td>RR036B</td>
			<td>RNase-free water (2) in kit</td>
		</tr>
		<tr>
			<td>Sterile 12&#34; long forceps</td>
			<td>F.S.T</td>
			<td>91100-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile fine forceps</td>
			<td>F.S.T</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile fine scissors</td>
			<td>F.S.T</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost PLUS Gold Slides</td>
			<td>Fisher</td>
			<td>1518848</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Fisher</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>MT25900CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X) phenol red</td>
			<td>Thermo Fisher</td>
			<td>12605010</td>
			<td>Cell-dissociation enzyme in the protocol</td>
		</tr>
		<tr>
			<td>Visium Accessory Kit</td>
			<td>10X Genomics</td>
			<td>PN-1000215</td>
			<td></td>
		</tr>
		<tr>
			<td>Visium Gateway Package, 2rxns</td>
			<td>10X Genomics</td>
			<td>PN-1000316</td>
			<td></td>
		</tr>
		<tr>
			<td>Visium Spatial Gene Expression Slide &#38; Reagent Kit, 4 rxns</td>
			<td>10X Genomics</td>
			<td>PN-1000184</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and profiling of human primary mesenteric arterial endothelial cells at the transcriptome level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The analysis of ECs from mesenteric artery using a combination of mechanical and enzymatical dissociation or cryopreservation for use in various downstream assays is depicted here (Figure 1). ECs can be profiled in mesenteric arteries using the following steps: A) mechanical dissociation from the intima coupled with collagenase digestion to culture cells; B) generation of single-cell suspension for scRNA-seq; or C) cross-sections of the artery can be embedded in OCT to be cryosectioned to pr...

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Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the ...

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2.8K Views.  City of Hope. The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Video: Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...