Endothelial induction

CLP‐CMCs (2x10⁴cells/cm²) were seeded on 24‐well plates coated with 0.5 ml of Matrigel (diluted 1:10 in standard medium) (BD Bioscience) and cultured in a humid atmosphere with 5% CO₂. At 3 and 7 days from induction, the gene expression of platelet endothelial cell adhesion molecule/CD31 was analysed by one‐step reverse transcriptase–PCR (Qiagen, Hilden, Germany). In parallel, we evaluated the formation of capillary‐like structures by optical microscopy using an inverted microscope Motic AE2000 (Motic^®, Wetzlar, Germany) equipped with Nikon DS‐L1 camera (Nikon, Düsseldorf, Germany), and the expression of blood‐clotting protein factor VIII/FVIII by immunofluorescence. Finally, to better explore the endothelial potential of CLP‐CMC cells, extracellular vesicles/exosomes (EVs/exs) were isolated using Cell Culture‐Nanovesicles kit (Biofield Innovation Srl, Padova, Italy) from conditioned media according to the manufacturer's protocol. After labelling with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma‐Aldrich), all samples were characterized by FCM for size, using as reference polystyrene beads supplied in Flow Cytometry Size Calibration Kit (Molecular Probes, Inc, Eugene, OR), and expression of CD9 or CD63 by indirect staining, according to the Pospichalova protocol 14. Moreover, we analysed by WB the expression of tetraspanin family protein/CD9, FVIII, Wnt3a ligand. Extracellular vesicles/exosomes from resting cells were considered as reference. For excluding cells from the analysis, cis‐Golgi marker/GM‐130 was considered as staining control. All antibodies used are listed in Table 2.