Name: isolation and profiling of human primary mesenteric arterial endothelial cells at the transcriptome level
Uploaded: 2022-03-14T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level at JoVE.com

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 ...

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...

Watch this Scientific Journal Video about Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level at JoVE.com

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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