Vegf165

A 3D spheroid-based angiogenesis assay was performed according to the procedure described by Heiss^{35 (link)} with a slight modification. After one passage, HUVECs were plated for siRNA or DNA transfection. Then, 24 h after transfection, cells were suspended in sterile 0.24% w/v methylcellulose solution in EGM2 medium with 10% FBS and aggregated overnight in hanging drops (25 μL) to form cellular spheroids (500 cells/spheroid). Spheroids were embedded into 1 mg/mL bovine acid-treated collagen gels (Nippi, Tokyo, Japan). Vessel sprouting was stimulated with 20 ng/mL (final concentration) VEGF165 (Sigma-Aldrich, MO, USA) 30 min after embedding the spheroids. After a 24 h incubation at 37 °C, spheroids in collagen gels were fixed with 10% paraformaldehyde and in vitro angiogenesis was quantified using cellSens Standard2 software (Olympus, Japan) to measure the cumulative sprout length (CSL) of capillaries, defined by the presence of lumens, that grew from the spheroids after 3 separate HUVEC transfections. When HUVECs or HDMECs were transfected with BOSβgal, whole-mount X-gal staining of spheroids was performed according to the protocol described in^{5 (link)}.

To initiate endothelial cell differentiation, 10,000 cells/cm² of passage 11–20 single cell suspension of iPSCs were seeded on matrigel (Corning) coated plates in mTeSR™1 media and incubated overnight to obtain 95% confluent culture. Once cells reached > 95% confluence, N2B27 medium supplemented with 8 µM CHIR-99021 and 25 ng/mL hBMP4 was added. Day 4–6, StemPro-34 medium supplemented with 300 ng/mL VEGF165 (sigma), 10 µM SB431542 and 2 µM forskolin. These endothelial cells were characterized for the expression of CD31 using flowcytometry and immunocytochemistry and were further evaluated for in vitro angiogenesis potential. These endothelial cells were found suitable for modeling vascularization of patient-derived organoids, gene expression analysis, and other downstream assays, that were performed.

Cell migration was assessed using a modified-Boyden chamber assay. CACs were detached from culture slides, washed with PBS and resuspended at a density of 5×10⁵/ml in migration medium (EBM-2 medium containing 0.5% BSA). 500 µl (2.5×10⁵) of cells were then added into the top chamber of the modified Boyden chamber apparatus (BD Biosciences, 8 µm pores). The chemoattractants VEGF165 (Sigma) or stromal cell derived factor 1 (SDF1, R&D Systems), used to promote migration, were prepared with the migration medium at concentrations of 50 ng/ml and 100 ng/ml, respectively. 500 µl of chemoattractant or migration medium alone was added to the lower chamber. Following a 5 hour incubation period at 37°C migratory, cells present on the underside of the insert were fixed and stained using Diff Quik (Fisher Scientific) and visualized by light microscopy. Images were acquired for 5 randomly selected fields and the mean number of cells from these fields was determined. Data are presented as fold change in cell migration towards chemoattractant compared to the respective control basal migration.

Pro-angiogenic agent was loaded into the central microchannel with the use of an automatic pipette. Firstly, VEGF₁₆₅ (Sigma-Aldrich, USA) with different concentration values (5, 25, 50, 100 ng/mL) was used. Every day, solution within a central microchannel was replaced by a fresh one. Cell culture within a microplatform was maintained for no longer than 10 days.