Fibroblast Growth Factor 2

An ALDEFLUOR assay kit (Stemcell Technologies, Vancouver, Canada) was used to sort ALDH^high CSCs from HN6 and SCC15 cells following the manufacturer’s protocol. Flow cytometry was used to detect green fluorescence-positive cells among live cells, in comparison with the fluorescence intensity of the diethylaminobenzaldehyde (DEAB) treated sample. These detected cells with high ALDH activity (ALDH^high cells) were used for subsequent experiments. Human HNSCC xenografted tumors were chopped into small pieces and digested into single-cell suspensions using a human tumor cell dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to isolate CSCs from human HNSCC PDX. An EpCAM-PE (Miltenyi Biotec) was used to isolate EpCAM⁺ tumor cells, and an ALDEFLUOR assay kit was used to sort the ALDH^high and ALDH^low subpopulations from the EpCAM⁺ tumor cells by flow cytometry. For the tumorsphere formation assay, ALDH^high and ALDH^low cells were added to ultralow attachment plates and cultured in DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) without serum but containing 1% B27 supplement (Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), human recombinant epidermal growth factor (EGF, 20 ng/mL, R&D Systems, Minneapolis, MN, USA), and human recombinant basic fibroblast growth factor (bFGF, 10 ng/mL, R&D Systems). After 10 days, spheres with a diameter exceeding 70 μm were counted under a microscope.

One-step digestion of skeletal muscle tissue for fibro-adipogenic progenitor isolation was performed as described before with few modifications (Contreras et al., 2020 (link)). Briefly, skeletal muscles from both hindlimbs of female wild type mice were carefully dissected, washed with ice-cold DMEM, and cut into small pieces with blades until a homogeneous, paste-like slurry was formed. Seven ml of digestion solution containing collagenase type II (265 Unit/mL, Worthington, DC, United States), 0.5 U of Dispase (Cat. No. 07913, STEMCELL™ Technologies, Canada), 0.05 mg/mL of DNaseI (Cat. No. 10104159001, Roche/Sigma-Aldrich, 100 mg from bovine), and 1% BSA (Sigma-Aldrich Pty Ltd., A3311-50G) dissolved in DMEM (Cat. No. 10566016) was added to two hindlimbs and the preparation was placed on a water bath with constant rotation at 37°C for 45 min and intermittent vortexing every 15 min. Tissue preparations were gently pipetted up and down 5–10 times to enhance muscle dissociation with a 10 mL Stripette^® serological pipette on ice. Ice-cold FACS buffer was added to make the final volume up to 30 mL volume and samples were then passed through 100 μm cell strainer sequentially after gentle mixing. Following centrifugation at 600 g for 6 min at 4°C, the pellet was resuspended in 10 mL of growth media (20 ng/mL of basic Fibroblast Growth Factor (Milteny Biotec, Cat. No. 130-093-843) and 10% heat-inactivated fetal bovine serum (v/v) (FBS; Hyclone, UT, United States) in DMEM (Cat. No. 10566016) and supplemented with antibiotics (Penicillin-Streptomycin Cat. no. 15140122, Gibco by Life Technologies) and cells were pre-plated onto 100 mm plastic tissue culture dish for 2 h and grown at 37°C in 5% CO₂ as previously described (Contreras et al., 2019a (link)). After 2 h of FAP pre-plating the supernatant media was removed to culture muscle stem cells (see Muscle stem cell enrichment and myotube differentiation protocol below) and replaced with fresh growth media. FAP CFU-F assay was performed with cells seeded at a density of 250/cm² in growth media in a 12-well plate coated with Corning Matrigel Matrix hESC qualified (Cat. No. 354277) prepared in cold DMEM/F-12 as per the provider’s instructions. Cultured FAPs were allowed to grow for about 7 days before splitting them. CFU-F experimental outline is shown in Figure 6B. FAPs were used not further than passage 1. CFU-F averages were obtained from three technical replicates/samples using three female mice. CFU-F photos were taken using an iPhone XR 12MP Wide camera.