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* These authors contributed equally
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 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.
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.
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' 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)
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.......
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.......
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 ....
Name | Company | Catalog Number | Comments |
1.5 mL micro-centrifuge tube | USA Scientific | 1615-5500 | |
10 cm dish | Genesee Scientific | 25-202 | |
23G needles | BD | 305145 | |
2-methylbutane | Thermo Fisher | AC327270010 | |
40 µm strainer | Fisher | 14100150 | |
4200 TapeStation System | Agilent Technologies | G2991BA | |
5 mL tube | Thermo Fisher | 14282300 | |
6-well plate | Greiner Bio-One | 07-000-208 | |
Attachment factor | Cell Applications | 123-500 | Attachment reagent in the protocol |
Black wax | Any commercial black wax can be used | ||
Bovine serum albumin heat shock treated | Fisher | BP1600-100 | |
CaCl2 | Fisher | BP510 | |
Centrifuge | Eppendorf | ||
Chloroform | Fisher | C607 | |
Collagenase D | Roche | 11088866001 | |
Cryostat | Leica | ||
Cryostat brushes | |||
D-Glucose | Fisher | D16-1 | |
Dimethyl sulfoxide | Fisher | MT25950CQC | |
Dispase II | Roche | 4942078001 | Bacteria-derived protease in the protocol |
Disposable Safety Scalpels | Myco Instrumentation | 6008TR-10 | |
D-PBS | Thermo Fisher | 14080055 | |
Ethanol | Fisher | BP2818-4 | |
Fetal bovine serum | Fisher | 10437028 | |
Hemocytometer | Fisher | 267110 | |
HEPES | Sigma Aldrich | H3375-100g | |
High sensitivity D1000 sample buffer | Agilent Technologies | 5067-5603 | |
High sensitivity D1000 screen tape | Agilent Technologies | 5067-5584 | |
Incubator | Kept at 37 °C 5% CO2 | ||
Isopropanol | Fisher | BP26324 | |
KCl | Fisher | P217-3 | |
Liquid nitrogen | |||
Medium 199 | Sigma Aldrich | M2520-10X | |
Metal cannister | |||
Microscope | Leica | To assess cell morphology | |
Microvascular endothelial culture medium | Cell Applications | 111-500 | |
NaCl | Fisher | S271-1 | |
New Brunswick Innova 44/44R Orbital shaker | Eppendorf | ||
Optimal Cutting Temperature compound | Fisher | 4585 | |
Plastic cryomolds | Fisher | 22363553 | |
RNA screen tape | Agilent Technologies | 5067-5576 | |
RNA screen Tape sample buffer | Agilent Technologies | 5067-5577 | |
RNase ZAP | Thermo Fisher | AM9780 | |
RNase-free water | Takara | RR036B | RNase-free water (2) in kit |
Sterile 12" long forceps | F.S.T | 91100-16 | |
Sterile fine forceps | F.S.T | 11050-10 | |
Sterile fine scissors | F.S.T | 14061-11 | |
Superfrost PLUS Gold Slides | Fisher | 1518848 | |
TRIzol reagent | Fisher | 15596018 | |
Trypan Blue | Corning | MT25900CI | |
TrypLE Express Enzyme (1X) phenol red | Thermo Fisher | 12605010 | Cell-dissociation enzyme in the protocol |
Visium Accessory Kit | 10X Genomics | PN-1000215 | |
Visium Gateway Package, 2rxns | 10X Genomics | PN-1000316 | |
Visium Spatial Gene Expression Slide & Reagent Kit, 4 rxns | 10X Genomics | PN-1000184 |
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