Fibroblast growth factor 2 (fgf2)

Levels of TNF alpha, VEGF-C (Thermo Fischer, Waltham, MA, USA), VEGF-A (PromoCell GmbH, Germany), angiostatin IL-6, plasminogen and FGF2 (Elabscience, Houston, TX, USA) were measured using an enzyme-linked immunoabsorbent assay kit (ELISA). The procedures were performed according to instruction of the manufacturer.

MUSE cells were grown on cover slides in MW24 plate and then fixed in 4% formaldehyde solution for 15 min at room temperature. We performed Duolink assay following the manufacturer's instructions (Sigma) using SSEA‐3 (IBL) and FGF2 (Elabscience) as primary antibody. Micrographs were taken under a fluorescence microscope (Leica). The percentage of positive cells was calculated by counting at least 300 cells in different microscope fields.

Conditioned media were collected at day 7 and levels of secreted bone morphogenetic protein-2 (BMP-2; Elabscience, Houston, TX, USA), bone morphogenetic protein-4 (BMP-4; Elabscience, Houston, TX, USA), vascular endothelial growth factor A (VEGF-A; Elabscience, Houston, TX, USA), and fibroblast growth factor-2 (FGF-2; Elabscience, Houston, TX, USA) were measured by immunoassay. For each of these proteins, a titration curve was designed with provided standards. Equal volumes (100 μL) of the collected culture media were immobilised on antibody-coated wells and immunoassays of the investigated proteins were developed according to the manufacturer’s instructions. Absorbance of the developed reactions was measured by a microplate reader (BioRad Laboratories, Hercules, CA, USA) at 490 nm.