Pdgf ab

USCs were isolated from the urine of 6 healthy adult donors with an average age of 22.67 ± 1.49 years old according to the methods described in previous studies [20 (link), 21 (link)]. Briefly, sterile midstream urine samples were collected and centrifuged, and the cell pellets were washed and resuspended for further culture. Primary medium was used for the first three days, and RE/MC medium was used for further proliferation culture. The primary medium was made up of a 1 : 1 mix of high-glucose DMEM (HyClone, Utah, USA) and Ham's F12 nutrient (Gibco, Grand Island, USA) supplemented with 10% FBS (Gibco, Australia), 1% antibiotic-antimycotic (Gibco, Grand Island, USA), and the components of the REGM SingleQuot kit (Lonza, Basel, Switzerland). The RE proliferation medium contained 500 ml of RE cell basal medium supplemented with the components of the REGM SingleQuot kit. The MC proliferation medium contained high-glucose DMEM supplemented with 10% FBS, 1% GlutaMAX (Gibco, Japan), 1% NEAA (Gibco, Grand Island, USA), 1% pen/strep (Gibco, Grand Island, USA), 5 ng/ml bFGF (PeproTech, Rocky Hill, USA), 5 ng/ml PDGF-AB (PeproTech, Rocky Hill, USA), and 5 ng/ml EGF (PeproTech, Rocky Hill, USA). The RE/MC medium was made up of a 1 : 1 mix of RE proliferation medium and MC proliferation medium. Cells from the P3 generation were used for downstream experiments.

PCLSs were generated as previously described [50 (link)]. Briefly, warm, low gelling temperature agarose (2% by weight, Sigma) in sterile DMEM/Ham’s F12 (Gibco) was infiltrated into mouse lung through trachea. After the trachea being ligated with thread to retain the agarose inside the lung, the whole lung was excised and cooled on ice for 10 min to allow gelling of the agarose. Then the separated lobes were cut with a vibratome (VT1000 S, Leica, Buffalo Grove, IL, USA) to a thickness of 300 μm. The PCLSs were cultivated in DMEM/Ham’s F12 (Gibco) with 0.1% FBS (Gibco), 100 μg/ml streptomycin, 100 U/ml penicillin, 0.25 μg/ml amphotericin B, and 10 mmol/l HEPES (Sigma Aldrich) at 37°C in a humidified 5% CO₂ atmosphere. For control cocktail (CC) or fibrosis cocktail (FC) treatment, 50 ng/ml rKL was added to serum-free medium 12 h after serum withdrawal. After 24 h of klotho pre-incubation, mouse PCLSs were incubated with CC or FC for another 48 h. Both the CC and FC were prepared as previously described [21 (link)] (28314802). In brief, FC contains 5 ng/ml TGF-β (PeproTech), 5 μM PDGF-AB (PeproTech), 10 ng/ml TNF-α (PeproTech), and 5 μM LPA (Cayman Chemical, Ann Arbor, MI, USA).

MSCs were cultivated in DMEM high glucose (Invitrogen Life Technologies, Darmstadt, Germany) with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany), 5 ng/ml transforming growth factor-β1 (TGF-β1; R&D Systems, Wiesbaden, Germany), 5 ng/ml platelet derived growth factor-AB (PDGF-AB; Peprotech, Hamburg, Germany) and 30 μM ascorbic acid (Sigma-Aldrich) for at least seven days to induce smooth muscle cell differentiation as recently reported [24 (link)]. The myogenic differentiation medium (‘Myo’) without the growth factors and ascorbic acid was used as a control medium (‘CM’).

Human brain microvascular ECs (ScienCell and PromoCell, isolated from adult or fetal human brains) were maintained in EC medium (ScienCell, supplemented with VEGF-A). All cells were used between passages 2 and 5. All cells were checked and showed no mycoplasma contamination. Cells were treated with recombinant human PDGF-AA (100 ng/ml, Peprotech, 100-13 A), PDGF-AB (100 ng/ml, Peprotech, 100-00AB), PDGF-BB (100 ng/ml, Peprotech, 100-14B), Ki8751 (3 nM, Bio-Techne, 228559-41-9), crenolanib (5 nM, ChemieTek, CP-868596), anti-VEGF antibody B20 (10 μg/ml, Genentech), anti-PDGF-AA (10 μg/ml, Millipore, 07-1436), anti-PDGF-BB (10 μg/ml, Millipore, 07-1437), or control rabbit IgG (10 µg/ml, Cell Signaling, 2729).