Dulbecco modified eagle medium (dmem)

To maximize the SVF yield from both sample sources and properly prepare cells for flow cytometry, we equally treated LA and MLA samples with 1% collagenase type I in Dulbecco’s Modified Eagle Medium (D-MEM) (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37 °C for 45 min. After 1:2 dilution with 2% fetal bovine serum (Biosera, Nuaille, France) in D-MEM (Sigma-Aldrich), samples were filtrated through a 100 µm-cell strainer (BD Falcon, Corning, NY, USA) and centrifuged at 300 g for 10 min at RT. Supernatants were discarded and cell pellet resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL, USA). After 10 min, samples were filtered through a 40 µm-cell strainer (BD Falcon, Corning, NY, USA), centrifuged at 300 g for 10 min at RT and the cell pellet resuspended in D-MEM (Sigma-Aldrich). The cells were counted on the Sysmex XT1800 counter (Sysmex, Kobe, Japan).

The previously-described adherent cells were detached and labeled with CD19, CD34, CD73, CD90, CD45 and CD105 fluorescent conjugated antibodies (Becton, Dickinson and Company, NJ, USA). After evaluation by assessing the percentages of CD19(−), CD34(−), CD73(+), CD90(+), CD45(−) and CD105(+) cells, results of evaluation are displayed in Supplementary Figure S1A. The adipogenic, osteogenic and chrondrogenic differentiation abilities of the cells were also assessed in Figure S1C. For the adipogenic assay, 10000 MSCs were seeded into 24-well plates (per well) and cultured in DMEM (Sigma) containing 10% FBS and 100 mm/L indometacin, 10 μg/mL insulin, 0.5 mmol/L IBMX and 100 nmol/L dexamethasone (all Sigma) for every 3 day, then the cells were then stained with Oil Red O. For the osteogenic assay, 5000 MSCs (per well) were seeded into 24-well plates and cultured in DMEM (Sigma, City, CA, USA) containing 10% FBS and 0.1 μmol/L dexamethasone, 50 μmol/L ascorbic acid and 10 mmol/L β-glycerophosphate (all Sigma). After 4 weeks, the cells were stained with alizarin red (Sigma).For the chrondrogenic assay, 10000 MSCs were seeded into 24-well plates (per well) and cultured in DMEM (Sigma) containing 10% FBS and 10 ng/mL TGF-β, 10 μg/mL insulin and 100 nmol/L dexamethasone (all Sigma) for every 3 day, then the cells were then stained with toluidine blue.

Brain MVEC (bMVEC) were isolated by incorporating and slightly modifying previously described methods [35 (link),36 (link),37 (link),38 (link)]. Brains of 6–8 wk old male C57BL6/J mice (4–6 animals per isolate), were excised and stored in serum-free DMEM (Sigma, St. Louis, MO, USA) on ice before surgical removal of the olfactory bulbs, cerebellum and mid-brain white matter. The remaining cortical tissue was rolled on sterile filter paper and subsequently digested in DMEM containing 2 mg/mL collagenase A (Roche, Basel, Switzerland) and 10 µg/mL DNase I (Roche) at 37 °C for 1 h, with gentle rotation. Digested tissue was pelleted at 290× g and resuspended in DMEM containing 20% BSA (Sigma) (w/v), then myelin fraction was separated by centrifugation at 1000× g for 10 min. The cell pellet was resuspended and filtered through a 70 µM nylon mesh and collected following centrifugation at 290× g. Cells were further digested in 2 mg/mL collagenase/dispase (Roche) and 10 µg/mL DNase I at 37 °C for 30 min. Following one wash in DMEM at 290× g, bMVEC were selected in medium supplemented with 4 µg/mL puromycin dihydrochloride (Sigma) [39 (link),40 (link)] for the first 4 days.

To maximize the cell yield from all three sample types and properly prepare cells for flow cytometry, we equally treated LA, MLA and SVF samples with 1% collagenase type I in Dulbecco’s Modified Eagle Medium (D-MEM) (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37 °C for 45 min. After 1:2 dilution with 10% heat-inactivated fetal bovine serum (Biosera, Nuaille, France) in D-MEM (Sigma-Aldrich), samples were filtrated through a 100 μm cell strainer (BD Falcon, Corning, NY, USA) and centrifuged at 300× g for 10 min at RT. Supernatants were discarded, and the cell pellet was resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL, USA). After 10 min, samples were filtered through a 40 μm-cell strainer (BD Falcon, Corning, NY, USA), centrifuged at 300× g for 10 min at RT and the cell pellet resuspended in D-MEM (Sigma-Aldrich). The cells were counted on the Sysmex XT1800 counter (Sysmex, Kobe, Japan), and sample volumes were adjusted to contain 3 × 10⁶ cell/mL.

H1299 cells (ATCC) were grown in DMEM + 10% FBS (Sigma or Gibco) and transfected by Lipofectamine 2000 (Invitrogen) - 3 μl of the reagent per 3 μg total plasmid DNA for a 12-well plate well at approx. 80% cell confluence (and proportionally for other vessel sizes, including Nunc 8-well LabTek chambers). H358 cells (ATCC) were grown in RPMI + 10% FBS (Gibco) and transfected 2x by Lipofectamine 2000 (Invitrogen) - 6 μl of the reagent per 5 μg total plasmid DNA for a 6-well plate well at approx. 80% cell confluence and for the second time 24h later. MEF cells (TP53 -/-, MDM2 -/-) were a kind gift of prof. U. Hibner (Institut de Génétique Moléculaire de Montpellier, France), were grown in DMEM + 10% FBS (Sigma) and were transfected by Effectene (Qiagen) according to the manufacturer's manual. MCF7, SkBr3 and MDA-MB-468 (ATCC) cells were grown in DMEM + 10% FBS (Sigma).