Chick embryo extract

MVSCs were isolated from rat aortic explants as described previously [5 (link)]. Briefly, male Sprague Dawley rats were first anesthetized with pentobarbital sodium (0.1 mg/g) and then perfused with 10 mL of PBS. Arterial tissues were harvested as quickly as possible and further dissected in DMEM supplemented with 1% fetal bovine serum (FBS). The aorta was then isolated from the lower thoracic aorta to the upper abdominal aorta. The endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blade before the adventitia was carefully removed from media following brief enzymatic digestion with 2.5 mg/mL of collagenase for 15 min at 37°C using forceps under a dissection microscope. The remaining media was cut into 1-mm pieces, placed onto the surface coated with 1% CellStart (Invitrogen) in 6-well plates and grown in MVSC culture medium containing DMEM with 2% chick embryo extract (MP Biomedical), 1% FBS, 1% N2 (Invitrogen), 2% B27 (Invitrogen), 100 nM retinoic acid (Sigma-Aldrich), 50 nM 2-mercaptoethanol (Sigma-Aldrich), 1% P/S and 20 ngml^-1 bFGF (R&D Systems) (maintenance medium). All procedures were approved by the University Animal Care Committee and were carried out in accordance with EU guidelines for the Protection of Animals used for Scientific Purposes, (Amendment) Regulations 2013 (S.I. No 434 of 2013).

Isolation of mouse heart cells and CD34⁺ cells was performed as previously described [29 (link), 30 (link)]. Then, cells were sorted with anti-CD34 magnetic beads (Miltenyi Biotec) according to manufacturer’s instructions. The cells were cultured as specified previously [31 (link)]. The purified CD34⁺ cells were seeded onto dishes with 0.1% gelatin (Sigma, G1393) and maintained in complete stem cell culture medium, which consists of DMEM, 1% FBS, 2% chick embryo extract (MP Biomedical), 100 nM retinoic acid (Sigma-Aldrich), 50 nM 2-mercaptoethanol (Sigma-Aldrich), 2% B27 (Invitrogen), 1% N2 (Invitrogen), 20 ng /ml bFGF (R&D Systems) and 1% P/S.
For cell differentiation experiments, CD34 + cells were cultured on gelatin-coated plates and maintained in complete stem cell culture with 50 ng/mL CTGF (Recombinant Human Connective Tissue Growth Factor, PeproTech, 120–19-20), which is sufficient to differentiate MSCs into fibroblast cells [32 (link)]. In the presence of recombinant TGFβ1 (5 ng/mL) (R&D systems, 7666-MB-005), cells were also treated with inhibitors including SB525334 (a selective TGFβ1 receptor inhibitor) (2 μM).

YFP-labeled MuSCs were isolated by FACS from control and cKO mice specified in text, figures, and legends. Briefly, hindlimb muscles were minced and digested with 0.2% collagenase (Worthington Biochemical) for 90 min followed by 0.2% dispase (Thermo Fisher Scientific) for 30 min in 37°C shaking water bath. Triturated muscle suspension was filtered through a 40 μm cell strainer (Corning) and subjected to isolation by BD FACSAriaIII. For culture, mononuclear cells were seeded on Matrigel (Corning) coated plates in growth media (DMEM with 20% FBS, 5% horse serum, 1% pen-strep, 1% glutamax (above from Gibco), 0.1% chick embryo extract (MPbio) and 2 ng/mL FGF2; R&D systems) for specified time in text and legends, before fixation and analysis.