Es 009 b

The aortic arch and root from C57BL/6 mice were harvested under sterile conditions as previously described [10 (link)]. Then, the tissue was sliced into 1 mm × 1 mm pieces and placed in a T25 flask previously coated with 0.1% gelatin (Sigma, G1393, USA). The T25 flask was inverted in a 5% CO₂, 37 °C incubator for 2–3 h until the tissue was attached to the flask. Subsequently, the flask was overturned and the complete stem cell medium was added. The medium, which comprised DMEM (American Type Culture Collection, 30-2002, USA), 10% ES cell qualified fetal bovine serum (Embriomax, ES-009-B, USA), 10 ng/ml of leukemia inhibitory factor (Merck Millipore, LIF1050, USA), 0.1 mM 2-mercaptoethanol (Sigma, M3148, USA) and 100 U/ml Penicillin–Streptomycin solution (Cienry, CR-15140, China), was changed every other day. Cells were passaged at a ratio of 1:5 when they reached 90% confluency. Then, cells were washed using PBS, detached using 0.05% trypsin–EDTA, resuspended in complete stem cell medium and seeded in 0.04% gelatin coated flasks.

Harvested tissue (20–50 ml) was washed in sterile PBS (10010; Gibco) containing 1 % penicillin and streptomycin (P/S, P4333; Sigma-Aldrich) and cleaned of any visible blood vessels and fibrotic layers, minced mechanically with sterile scissors, and digested with 0.01 % collagenase IV (17104-019; Life Technologies) DMEM F12 (SH3027101; HyClone) solution for 2 h at 37 °C. The acquired cell suspension was centrifuged at 300 × g for 5 minutes. The supernatant was discarded and a stromal vascular fraction pellet was collected, washed, and filtered (100 μm and 40 μm cell strainer). The achieved cell fraction was plated onto cell culture flasks and cultured in low-glucose DMEM media (11885; Gibco) supplemented with 10 % FBS (ES-009-B; Millipore) and 1 % P/S in 5 % CO₂ at 37 °C. Cultures were washed after 72 h to remove unattached cells and expansion medium was changed every 48 h thereafter. To keep cells at low density, preventing cell death and spontaneous differentiation, cultures were repassaged when reaching 80 % confluence. Cells were harvested using 0.25 % trypsin (TrypLE™; Life Technologies) for 3 min at 37 °C, followed by trypsin inactivation using 2 volumes of culture medium containing serum and replated onto new cell culture dishes/flasks at a seeding density of 3 × 10⁴ per 75 cm² culture flask.

The non-polarized or M0 phenotype was achieved by cultivating cells in RPMI-1640 medium supplemented with 10% FBS (Cat. No. ES-009-B; Millipore, Burlington, MA, USA), 10% MCSF (L929-CM), 1% PEST, and 1% L-glutamine for 72 h. The media was supplemented either with 50 ng/mL recombinant mouse IFNγ (Cat. No. BMS326; eBioscience, San Diego, CA, USA) and 100 ng/mL Pam3SCK4 (Cat. No. tlrl-pms; InvivoGen, San Diego, CA, USA) to achieve M1 phenotype, or with 20 ng/mL IL-4 (Cat. No. RP-8666; Invitrogen, Waltham, MA, USA) to achieve M2 phenotype.

In vitro culture (IVC) assay of mouse embryos was carried out according to the procedures that we previously described (52 (link)). Briefly, the mouse blastocysts (E3.5) were recovered from the mating of Cnot8 heterozygote females and males, transferred to 3.5 cm petri dishes precoated with Matrigel (BD, BD356230), and cultured in CMRL 1066 medium (Gibco, 11530037) containing 10% FBS (Millipore, ES-009-B), 1 mM sodium pyruvate, 100 units/ml penicillin-streptomycin, 2 mM L-Glutamine (Gibco, 25030081) at 37°C under 5% CO₂ for the first two days (IVC day 1–2). When the embryos were solidly attached onto the petri dishes, the culture medium contained 20% FBS was used. After culture for additional 2–3 days, the embryos developed into egg cylinder-like stage were used for the next experiments. For genotyping the IVC embryos, nested PCR and Sanger sequencing were used. All primers used were listed in Supplementary Table S1.

A single cell suspension of analyzed cells was collected by treating the cells with 0.25 % trypsin for 3 min at 37 °C, followed by washing in complete culture medium. Cells were washed twice with 3 % FCS–PBS (ES-009-B; Millipore), resuspended at a concentration of 5 × 10⁵ cells/ml, and stained with allophycocyanin, fluorescein isothiocyanate, phycoerythrin, Alexa 647, or PerCP/Cy5.5-conjugated monoclonal mouse-anti-human CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, and CD166 antibodies and lineage cocktail 1 (lin-1; BD Biosciences: CD3, CD14, CD16, CD19, CD20, CD56) at 4 °C for 30 min. Cells were then washed twice, resuspended, and analyzed for cell surface marker expression by flow cytometry (FACSCalibur™; BD Biosciences).