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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

This laboratory previously demonstrated that induction of d-flow leads to quick and rugged atherosclerosis development in hyperlipidemic mice^1,2. The novel mouse model of d-flow-induced atherosclerosis was possible using partial carotid ligation (PCL) surgery ³. PCL surgery induces low and oscillatory blood flow condition or d-flow in the ligated left carotid artery (LCA). In contrast, the contralateral right carotid artery (RCA) continues to face stable laminar flow (s-flow). Previously, to understand the effect of d-flow on endothelial cells, the carotid arteries were dissected out after partial ligation surgery and flushed with a phenol and guanidine isothiocyanate-based lysing agent (luminal RNA/DNA flushing method)^2,4, which provided endothelial-enriched "pooled bulk" RNAs or DNAs. These pooled bulk RNAs or DNAs were then processed for transcriptomic studies or epigenomic DNA methylome studies, respectively^4,5,6. These studies helped discover multiple flow-sensitive genes and microRNAs whose roles in endothelial biology and atherosclerosis were extensively investigated^4,6,7.

However, despite endothelial enrichment, these bulk RNA/DNA studies could not distinguish the specific role of each cell type in the artery wall in d-flow-induced atherosclerosis. Endothelial-enriched single-cell (sc) isolation and scRNA and scATAC sequencing studies were performed to overcome this limitation⁸. For this, C57Bl6 mice were subjected to the PCL surgery to induce d-flow in the LCA while using the s-flow-exposed RCA as control. Two days or two weeks after the PCL surgery, the mice were sacrificed, and the carotids were dissected and cleaned up. The lumen of both LCAs and RCAs were infused with collagenase, and the luminal collagenase digests containing ECs as a significant fraction and other arterial cells were collected. The single-cell suspension (scRNAseq) or single-nuclei suspension (scATACseq) were prepared and barcoded with unique identifiers for each cell or nucleus using a 10x Genomics setup. The RNAs were subjected to cDNA library preparation and sequenced.

The scRNAseq and scATACseq datasets were processed using the Cell Ranger Single-Cell Software and further analyzed by Seurat and Signac R packages^9,10. Each cell and nucleus was assigned a cell type from these analyses and clustered into the cell type based on the marker genes and unique gene expression patterns. The results of the scRNAseq and scATACseq demonstrated that these single-cell preparations are enriched with ECs and also contain smooth muscle cells (SMCs), fibroblasts, and immune cells.

Further analysis revealed that the EC population in the luminal digestion is highly divergent and plastic (8 different EC clusters) and responsive to blood flow. Most importantly, these results demonstrated that d-flow reprograms ECs from an athero-protected anti-inflammatory phenotype to pro-atherogenic phenotypes, including pro-inflammatory, endothelial-to-mesenchymal transition, endothelial stem/progenitor cell transition, and most surprisingly, endothelial-to-immune cell-like transition. In addition, scATACseq data reveal novel flow-dependent chromatin accessibility changes and transcription factor binding sites in a genome-wide manner, which form the basis of several new hypotheses. The methodology and protocol for preparing single endothelial cells for single-cell multi-omics studies from the mouse carotid arteries are detailed below.

Protokół

All animal procedures described below were approved by the Institutional Animal Care and Use Committee at Emory University. Non-hypercholesterolemic, age- and sex-matched C57BL/6 mice were used to mitigate sex-dependent variation and offset any complication of hypercholesterolemic conditions.

1. Partial carotid ligation (PCL) surgery

NOTE: Partial carotid artery ligation of LCA was carried out as previously described and demonstrated³.

1. Anesthetize the mouse by isoflurane inhalation (5% isofluorane in oxygen for induction and 1.5% after that for maintenance) throughout the procedure using an anesthetic vaporizer. Place the mouse supine on a Deltaphase isothermal pad to prevent loss of body heat during surgery.
2. Apply an ocular ointment to prevent exposure keratitis, and confirm the depth of anesthesia by the absence of toe pinch response.
3. Inject buprenorphine 0.05 mg/kg subcutaneously to manage pain preemptively. Disinfect the epilated area by applying and cleaning with betadine and isopropanol solution three times. Ensure that betadine is the last step to sterilize the surgery area, starting from the center and finishing at the sides.
4. Using aseptic techniques, make a ventral midline incision (~1 cm) in the neck region, and expose the LCA branch point by blunt dissection.
5. Ligate the left internal carotid, external carotid, and occipital arteries with 6-0 silk suture; leave the superior thyroid artery untouched.
6. Approximate the skin and close the incision with tissue glue and/or sutures.
7. Transfer the mouse to a pre-warmed recovery cage (maintained at 37 °C) and place it on a clean towel for up to 1 h to avoid post-surgery hypothermia.
   NOTE: In our experience, a pre-emptive single dose of Buprenorphine (0.05 mg/kg) is usually sufficient for post-surgery pain management. A repeat dose of Buprenorphine can be administered if the animal is in distress after the first 24 hours. If performed correctly, there is no mortality associated with this procedure, and postoperative stress to the mouse is minimal. The mice are returned to their respective cages and monitored daily following the surgery.
8. Prepare the perfusion setup.
   1. Use 0.9% NaCl (normal saline) containing 10 U/mL heparin in an IV bag. Hook the IV bag 244-274 cm (8-9 feet) from the ground or approximately 122-152 cm (4-5 feet) higher than the dissection board. Attach the butterfly needle to the end of the saline line and flush any air bubbles out of the IV line.

2. Isolation of carotid arteries post sacrifice

1. Sacrifice the mice by CO₂ inhalation following the institutional IACUC protocol.
2. Spray 70% ethanol on the mouse skin and place it in the supine position on the dissection board containing adsorbent paper towels. Secure the paws of the mouse to the adsorbent towels on the dissection board using adhesive tape or a 21 G needle.
3. Using a pair of sterile scissors, remove the skin of the mouse starting from the abdomen and all the way to the top of the thorax.
4. Use a pair of scissors to open the abdominal wall below the ribcage by blunt dissection.
5. Using the scissors, carefully make an incision along the length of the diaphragm, and continue through the ribs on both sides of the thorax until the sternum can be lifted away. Gently lift away the sternum with a pair of forceps; after that, remove the ribcage to expose the heart.
6. Carefully remove the thymus and any connective tissue over the heart to visualize the major vessels.
7. Sever the vena cava with scissors to allow blood to exit from closed circulation.
8. Insert a 21 G butterfly needle connected to the IV line through the apex of the heart into the left ventricle and allow retrograde perfusion for 2-3 min with normal saline at room temperature. Ensure that a constant flow rate of normal saline is maintained by keeping the saline bag at the height of 8-9 feet from the ground.
   NOTE: The lungs and liver become pale, indicating optimal perfusion. Approximately 20 mL of perfusion buffer is used for each mouse.
9. Remove the skin from the neck region and remove all the fat, muscles, and connective tissues to expose the carotid arteries (Figure 1A).
   NOTE: The rest of the procedure is carried out under the dissecting microscope.
10. Adjust the field of view to locate the ligation sites. Using the 10 mm micro-dissection scissors, make a small incision below the ligation site in the LCA to allow perfusion.
11. Perfuse again for an additional 1 min via the left ventricle and ensure that the LCA is well perfused and free from any visible traces of blood.
12. Use fine-tip forceps and small spring scissors to carefully remove the peri-adventitial tissues surrounding the carotids while the carotid is attached to the body.
    NOTE: Be extremely careful not to squeeze or stretch the carotid arteries during this cleaning step, as this can increase the number of non-viable cells. If performed correctly, the cell viability in the final cell suspension is usually >93%.

3. Endothelial-cell-enriched single-cell isolation from mice carotids

NOTE: The reagents described below can be prepared in advance and stored at 4 °C until use: 1x and 0.1x single-nuclei lysis buffers with the reagents listed in Table 1; single-nuclei wash buffer with the reagents listed in Table 1; single-nuclei Nuclei Buffer recipe with the reagents listed in Table 1. The working stocks of these buffers are to be prepared following the manufacturer's protocol. Digestion buffer composition: Collagenase Type II 600 U/mL and DNase I 60 U/mL in 0.5% fetal bovine serum (FBS)-containing phosphate-buffered saline (PBS).

1. While the carotids are still attached, wash the exterior of the carotid arteries with normal saline solution to flush away any traces of blood.
2. Using an insulin syringe fitted with a 29 G needle, inject ~ 50 µL of the digestion buffer into the lumen of the distal end of the left carotid artery. As the digestion buffer starts to fill the carotid artery, clamp the proximal end of the carotid with a micro-clip (Figure 1B). Introduce an additional 15-20 µL of digestion buffer into the lumen and clip the distal end to avoid the release of the digestion buffer.
3. Carefully explant the carotid arteries from the mouse (Figure 1B,C) and place them in 35 mm dishes containing warm (at 37 °C) Hepes-buffered saline solution.
   NOTE: Make sure that both the clamps are securely placed. If the clamp becomes loose, the digestion solution can leak out of the lumen and affect the obtained cell count. If this happens, add additional enzymatic solution using an insulin syringe fitted with a 29 G needle from the open end of the carotid and secure the clamp again (Figure 1D). If the enzyme buffer solution leaks again, it is likely that the carotid is severed during the explantation process. In this case, discard the carotid and exclude the sample from the study. Repeated rough handling of the carotid will decrease the cell viability and the quality of single-cell preparation. Take care to identify and correctly label the left and right carotid arteries.
4. Incubate the explanted carotids for 45 min at 37 °C with intermittent rocking.

4. Flushing the carotid arteries

1. After completing the luminal enzymatic digestion, remove the carotid artery together with the clamps from the 35 mm dish. Carefully remove each clamp, taking care that the digestion buffer should not leak.
2. Gently hold one end of the carotid artery with fine forceps on top of a 1.5 mL microcentrifuge tube. Insert a 29 G needle fitted with an insulin syringe containing 100 µL of warm digestion buffer (37 °C) into the lumen with the other hand (Figure 1D).
3. Quickly flush the lumen of the carotid into the microcentrifuge tube. Block the enzymatic reaction by adding 0.3 mL of FBS into the 1.5 mL tube. Place the tube on ice.
   NOTE: The flushing contains the cells from the luminal enzymatic digestion. Adding FBS and reducing the temperature stops the enzymatic digestion process. If the number of cells in the preparation is high and contains debris or fat tissue, the use of a 50 or 70 µm cell strainer is highly recommended to filter out unwanted tissues debris. To increase the single-cell count, flushing multiple carotids is recommended. Here, to obtain at least 5,000 cells, 10-12 carotids were flushed and pooled as one sample.
4. Centrifuge the cells at 500 × g for 5 min at 4 °C using a centrifuge equipped with a fixed-angle rotor (see the Table of Materials).
5. Discard the supernatant and resuspend the cell pellet in digestion buffer containing cell dissociation reagent (see the Table of Materials) for 5 min at 37 °C to separate all the cells into single cells.
   NOTE: If the number of cells in the preparation is low, increase the centrifuge speed up to 2,000-3,000 × g to spin down the cells. Moreover, using 0.5 mL centrifuge tubes facilitates better visualization of the cell pellet at the bottom of the tube.
6. Block the enzymatic reaction by adding 0.15 mL of FBS into the 0.5 mL tube.
7. Centrifuge the single-cell suspension at 500 × g for 5 min at 4 °C using a centrifuge equipped with a fixed-angle rotor, as in step 4.4.
8. Discard the supernatant and resuspend the cells in 100 µL of ice-cold 1% bovine serum albumin (BSA) solution in PBS in a 0.2 mL microcentrifuge tube.
   NOTE: Using 0.2 mL centrifuge tubes enhances the visualization of the single-cell-pellet at the bottom of the tube.

5. Single-cell and single-nucleus analyses

1. Resuspend and submit the single-cell preparation for scRNAseq.
   1. Resuspend the single-cell pellet with 100 µL of ice-cold 1% BSA in PBS in a 0.2 mL tube.
   2. After resuspending the cells for single-cell encapsulation, immediately proceed for single-cell encapsulation and barcoding using a microfluidics-based single-cell partitioning and barcoding system (see the Table of Materials).
   3. Before submitting the samples to the Genomics Core, count and inspect the single-cell preparations; ensure the absence of cell aggregates.
      ​NOTE: The aggregation of single cells can be further minimized by increasing the BSA amount up to 2%.
2. Resuspend and submit the single-cell preparation for scATACseq.
   1. Resuspend the cell pellet in 100 µL of 0.04% BSA in ice-cold 1x PBS and centrifuge the single-cell suspension at 500 × g at 4 °C.
   2. Lyse the single-cell suspension with ice-cold 0.1x lysis buffer (Table 1) and incubate for 5 min on ice.
   3. Mix the lysate 10 times with a P-20 pipette and incubate for an additional 10 min.
   4. Add 500 µL of chilled wash buffer (Table 1) to the lysed cells and mix 5 times using a pipette.
      NOTE: Use a 70 µm cell strainer to filter any debris from the cell suspension and transfer them into a 2 mL tube.
   5. Centrifuge the lysate at 500 × g for 5 min at 4 °C. Discard the supernatant and resuspend the nuclei-pellet in the diluted nuclei buffer (150 µL) (see the Table of Materials and Table 1). Count and inspect the single-nuclei preparations using a hemocytometer¹¹.
   6. Submit the single-nuclei sample to the Genomics Core for single-nuclei transposition, nuclei partitioning, library preparation, and sequencing.
      NOTE: If the initial cell number is low, it is common to observe debris along the nuclei. Therefore, it is recommended to start with a high cell number.

Wyniki

Partial carotid ligation surgeries were performed on 44 mice, and the onset of d-flow in the LCA was validated by performing ultrasonography one day post partial ligation surgery. Successful partial ligation surgery causes reduced blood flow velocity and reverses blood flow (disturbed flow) in the LCA³. The carotid arteries were dissected out either at two days or at two weeks post ligation. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single-...

Dyskusje

This paper provides a detailed protocol to isolate single-cell preparations from the mouse carotid arteries. The influence of d-flow on the endothelial cells can be accurately studied if the PCL surgery is performed correctly. It is crucial to correctly identify the branches of the common carotid, such as the external carotid, internal carotid, occipital artery, and superior thyroid artery. Validation of flow patterns by ultrasonography further validates the successful onset of d-flow conditions. Althou...

Materiały

Name	Company	Catalog Number	Comments
Chemicals, Peptides, and Recombinant Proteins
1x PBS (Cell Culture Grade)	Corning	21040CMX12
1.5 mL Protein LoBind Microcentrifuge Tubes	Eppendorf	022-43-108-1
15 mL Centrifuge Tube - Foam Rack, Sterile	Fablab	FL4022
50 mL SuperClear Centrifuge Tubes	Labcon	3191-335-028
6-0 Silk Suture Sterile	Covidien	s-1172 c2
70 µm Cell Strainer, White, Sterile, Individually Packaged	Thermo Fisher Scientic	08-771-2
Accutase solution,sterile-filtered	Sigma-Aldrich	A6964-100ML	or equivalent
ATAC Buffer (Component I of Transposition Mix)	10x Genomics	2000122
ATAC Enzyme (Component II of Transposition Mix)	10x Genomics	2000123/ 2000138
Bovine Serum albumin	Sigma-Aldrich	A7906-500G
Buprenorphine	Med-Vet International	RXBUPRENOR5-V
Chromium Controller & Next GEM Accessory Kit	10X Genomics	1000204
Chromium Next GEM Single Cell 3' Reagent Kits v3.1	10X Genomics	1000121
Chromium Next GEM Single Cell ATAC Reagent Kits v1.1	10X Genomics	1000175
Collagenase II	MP Biomedicals	2100502.5
Digitonin	Sigma-Aldrich	D141-100MG
Dissecting Forceps	Roboz Surgical Instruments Co	RS-5005
Dnase1	New England Biolabs Inc	M0303S
Centrifuge (Benchtop-Model # 5425)	Eppendorf	22620444230VR
Fetal Bovine Serum - Premium Select	R&D systems	S11550
Fixed Angle Rotor	Eppendorf	FA-45-24-11-Kit Rotor
HEPES buffered saline	Millipore Sigma	51558
Insulin syringe (3/10 mL 29 G syringe)	BD	305932
Isoflurane	Patterson vet	789 313 89
MACs Smart Strainers (30 µm)	Miltenyi Biotec	130-098-458
MACS SmartStrainers (100 µm)	Miltenyi Biotec	130-098-463
Normal Saline (0.9% sodium chloride)	Baxter International Inc	2B1323
Nuclei Buffer (20x)	10x Genomics	PN 2000153/2000207
PBS (10x), pH 7.4	Thermo Fisher Scientic	70011-044
Small scissors	Roboz Surgical Instruments Co	RS-5675
Stainless Steel Micro Clip Applying Forceps With Lock	Roboz Surgical Instruments Co.	RS-5480	or similar
Tissue Mend II	Webster Veteinanry	07-856-7946
Type II Collagenase	MP biomedicals	2100502.1
Deposited Data
scATACseq FastQ files	NCBI	www.ncbi.nlm.nih.gov/bioproject Accession # PRJNA646233
scRNAseq FastQ files	NCBI	www.ncbi.nlm.nih.gov/bioproject Accession # PRJNA646233
Software and Algorithms
Cell Ranger 3.1.0	10X Genomics	https://support.10xgenomics.com/ single-cell-gene-exp
Cicero	Pliner et al., 2018	https://cole-trapnell-lab.github.io/cicero-release/
Ggplot2 v3.2.1	Hadley Wickham	https://cran.r-project.org
Harmony	Korsunsky et al., 2019	https://github.com/immunogenomics/harmony
ImageJ	Schneider et al., 2012	https://imagej.nih.gov
Monocle 2.8.0	Qiu et al., 2017	https://github.com/cole-trapnell-lab/ monocle-release
R version 3.6.2	R Foundation	https://www.r-project.org
Seurat 3.1.3	Stuart et al., 2019	https://github.com/satijalab/seurat
Signac 0.2.5	Stuart et al., 2019	https://github.com/timoast/signac