Fgf 2

To examine the effect of short-term treatment with CM, iopamidol (300 mgI/mL) was added to the luminal (upper) side of the E00 model. Following 30 min of incubation, CM was replaced with RBEC medium. Then, barrier function was monitored by measuring TEER for 24 h. Permeability of Na-F through the E00 models was measured at 6 h after replacing treatment with RBEC medium. To examine the dose- and time-dependent effects of iopamidol on the barrier function, iopamidol (0, 3, 15, and 30 mgI/mL) was added to the abluminal (lower) side of the E00 model. As the hyperosmolarity of Iopamidol may affect BBB function, mannitol (62 mM), which has the same osmolality as iopamidol (30 mgI/mL), was used as an osmotic control. Then, barrier function was monitored by measuring TEER for up to 72 h. To examine the effect of fibroblast growth factor-2 (FGF-2, ReproCELL Inc., city, Japan) on barrier dysfunction induced by iopamidol, iopamidol (30 mgI/mL) and/or FGF-2 (10 ng/mL) for 24 h was added to the luminal side of the E00 model. The effects of iopamidol on pericytes and astrocytes were examined by using the E00 and EPA models. Iopamidol (30 mgI/mL) was added to the abluminal side of these models.

Induction of differentiation of iPSC to EC was done by a modification of the previously reported method^{30 (link), 31 (link)}. Feeder-free iPSC were subcultured in 6-well plates at a density of 0.2 × 10⁵ cells/well on day −2. Differentiation induction medium was prepared with Advanced RPMI 1640^® (Thermo Fisher), 2% B-27 Minus Insulin^® (Gibco), 200 mM L-Glutamine (Gibco), 10 ng/mL of fibroblast growth factor (FGF2, ReproCell), 50 ng/mL of VEGF A-165 (Wako Co. Ltd., Japan), and 2 ng/mL of bone morphogenetic protein 4 (BMP-4, R&D). The standard culture medium was changed to induction medium with 1% matrigel (Corning) on day 0. Induction medium was exchanged every two days, and differentiation was completed on day 5 (Fig. 1). After sorting of EC (iEC) and MC by FACS analysis, only iEC were subjected to following subculture.