H1168

SC-derived myoblasts were transfected with selected miRNAs or inhibitors as described above. After 48 h of transfection, the culture medium was changed to Complete Human Endothelial Cell Medium (CHECM, H-1168, Cell Biologics) and the cells were cultured for 24 h. Primary human skeletal muscle microvascular endothelial cells (PMECs; H-6220, CellBiologics) were seeded in culture plates covered with a thick layer of Matrigel GFR Basement Membrane Matrix in the density of 35,000 cells/cm². The PMECs were cultured for 18 h either in Complete Human Endothelial Cell Medium or Complete Human Endothelial Cell Medium collected from control or transfected SC-derived myoblast cultures (SC-derived myoblasts conditioned medium). After 18 h of culture, the PMECs were fixed with 3% PFA. The number of branching points (nods) and the number of tubes formed in culture were calculated from at least 10 random fields of view for each variant of the experiment. Moreover, to evaluate the role of VEGF in tubulogenesis PMEC were cultured either in Complete Human Endothelial Cell Medium (CHECM) or Complete Human Endothelial Cell Medium collected from control or transfected SC-derived myoblast cultures (SC-derived myoblasts conditioned medium) in presence of 20 μg/ml anti-VEGF antibody (SP07-01, Invitrogen) for 16 h and fixed with 3% PFA.

Primary human brain microvascular endothelial cells (HBEC) purchased from Cell Biologics (H-6023, Chicago, IL, USA) were cultured on gelatin-coated plates with complete human endothelial cell medium (Cell Biologics H1168, Chicago, IL, USA) in a 5% CO₂ humidified incubator at 37 °C. All cells were plated at the same density prior to treatment. HBEC were treated with treated with 40 μg/mL tau oligomers (O. Tau), 40 μg/mL monomeric tau (M. tau), 40 μg/mL recombinant human cytokeratin-8 (KRT8, RayBiotech, Norcross, GA, USA), or vehicle (PBS) in 1 mL of complete media in a 35 mm plate for 42 h on a rocker (15 RPM) to simulate bidirectional low wall shear stress.