Human fgf 2

The muscle fragment was collected in mesoangioblast culture medium: IMDM medium containing 10%FBS (Bodinco), 0.1% gentamycin, 1X glutamine, 1X sodium pyruvate, 0.2% 2-mercapto ethanol, 1x Insulin-Transferrin-Selenium, 1X Non-Essential Amino Acids, and 5 ng/ml human FGF-2 (Miltenyi Biotec). All materials were obtained from Thermo Scientific, unless stated otherwise. Mesoangioblasts from a vastus lateralis skeletal muscle biopsy were isolated and cultured as described before [27 ]. In brief, skeletal muscle biopsies were rinsed with PBS, cut in small fragments, and plated on a type I collagen-coated dish with a few drops of the aforementioned medium. From the muscle biopsy, fibroblasts spread out while mesoangioblasts poorly adhere to these fibroblasts. After 10–14 days during which medium was regularly added, the medium containing mesoangioblasts was transferred to a new dish (5000/cm²) and MABs were cultured as attaching cells. Alternatively, outgrowth of the muscle biopsies was trypsinized after 10–14 days and seeded to a new dish at a 10,000 cells/cm². The following 2 days, the medium containing mesoangioblasts was transferred to a new dish (5000/cm²) and MABs were further cultured as attaching cells.

PBS (80 μL) or human Fgf2 (50 μg/mL) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, total dose 135 μg/kg [Yan et al. 2000 (link)]) were subcutaneously injected 30 min and every second day after SCI. The first injection was delivered at the back under the skin of the operated area just above the lesion site, whereas the other injections were performed subcutaneously at the abdominal area next to the left hind limb, where the secession is impaired in order to prevent unnecessary pain for the animal.

For single myofiber isolation, the EDL muscle was dissected tendon to tendon from 6- to 8-weeks-old mice and enzymatically digested in DMEM medium supplemented with 0,2% Collagenase I (#C0130, Merck KGaA) at 37 °C for 1 h. Post digestion, single myofibers were released by trituration with heat-polished glass Pasteur pipettes in manipulation medium (DMEM, 2% FBS, 1% Penicillin/Streptomycin). Floating fibers were fixed in 4% PFA after isolation (day 0, d0) or cultured in 2.0 ml tubes with no conical bottom (Eppendorf) in growth medium (40% DMEM, 40% HAM’s F12, 20% FBS, 1% Penicillin/Streptomycin, 2.5 ng/ml human FGF-2 (#130-104-924 Miltenyi Biotec) for 1 (d1), 2 (d2) and 3 days (d3).

MCC tumor samples were obtained from patient biopsies or mouse PDXs, which were generated as previously described (66 (link)). The tissue was minced manually, suspended in 2 mg/mL collagenase I (Sigma-Aldrich), 2 mg/mL hyaluronidase (Sigma-Aldrich), and 25 μg/mL DNase I (Roche Life Sciences), and incubated on a low-speed orbital shaker for 30 minutes. After digestion, the single-cell suspension was passed through a 100 μm strainer, washed, and cultured in NeuroCult NS-A Human Proliferation Kit (Stemcell Technologies) supplemented with 0.02% heparin (Stemcell Technologies), 20 ng/mL human EGF (Miltenyi Biotec), and 20 ng/mL human FGF-2 (Miltenyi Biotec). Established cell lines were tested as mycoplasma free (Venor GeM Mycoplasma Detection Kit, Sigma-Aldrich). Cell lines were authenticated as MCC through IHC for CK20 and SOX2 (Figure 1A and Supplemental Figure 1C), and as derivatives of original tumors by HLA typing, which was available for 7 of the 11 lines (Supplemental Table 6). Cell line sexes are described in Table 1. MKL-1 and WaGa lines were gifts from James A. DeCaprio’s laboratory and were grown in RPMI 1640 with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco).

5000 cells/mL were seeded in non-adherent bacterial plates with CSC medium (DMEM-F12 without L-Glutamine nor Hepes (Biowest, Nuaillé, France) supplemented with 1% Penicillin/Streptomycin (Biowest, Nuaillé, France), B-27™ (40 mL/L, ThermoFisher Scientific, Waltham, MA, USA), human FGF-2 (0.01 µg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and human EGF (0.02 µg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany). Medium was renewed weekly by low speed centrifugation (5 min, 800 rpm) until primary spheres were obtained after 3 weeks. To generate secondary spheres, primary spheres were disaggregated through a 29G needle and seeded again at a density of 5000 cells/mL in 60 mm suspension culture dishes (Corning, Corning, NY, USA). For assays using recombinant human ADAMTS1 (rhATS1, 2197-AD, R&D), spheres were grown in 6-well ultralow attachment plates (Corning, Corning, NY, USA), using CSC medium supplemented with 1 µg/mL rhATS1. For assays using conditioned medium (CM), fresh medium was collected from 24 h cultured MUM-2B cells and supplemented with B-27, FGF-2 and EGF as described above. Images were captured with an Axio Vert microscope (A-Plan 5x/0.12 objective, Zeiss, Oberkochen, Germany), and evaluated with Carl Zeiss ZEN 2.3 SP1 (black) software (Oberkochen, Germany). Sphere volume was calculated as: sphere volume = (π × length × width²)/6.