Evaluation of nintedanib activity on iPSC-derived endothelial cells. (A) Endothelial differentiation for KIT D816V or control iPSCs. Representative phase-contrast microscopy images of days 1, 7, 11, and 14 are shown. Scale bars, 500 μm. (B) KIT D816V and control iPSC–derived endothelial cells express CD34, CD31, CD105, and CD144 endothelial markers but lack CD45 and CD43 expression. (C) KIT D816V endothelial cells (red) have lower surface KIT receptor expression compared with control cells (blue), as shown by representative graphs; isotype control is shown in gray (n = 3) *P = .0005. (D) Immunofluorescence staining of KIT D816V and control endothelial cells showed homogenous surface expression of CD31 (left panels) and CD144 (middle panels). Cells effectively absorbed Dil-conjugated acetylated LDL (right panels). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 μm. (E) Quantitative RT-PCR data show similar expression of CD31, CD34, and CD144 in KIT D816V and control endothelial cells. KIT mRNA expression was slightly higher in mutated cells (n₌ 4-5). *P = .03. (F) KIT D816V and control endothelial cells derived from patients 1 and 3 iPSCs showed similar nintedanib response curves (left panel; n = 2-3). Patient 1 (P1), IC50_D816V = 1632 nM and IC50_control = 7003 nM; patient 3 (P3), IC50_D816V = 2073 nM and IC50_control=3803 nM). KIT D816V endothelial cells were more affected by 1 µM nintedanib treatment than were unmutated cells (right panel; n = 3-4). *P < .01, Welch’s t test. (G) Comparison of drug response curves obtained for KIT D816V (left panel) or control (right panel) endothelial cells (n = 2-3), HPCs (from Figure 3), and MCs (from Figure 4) treated with nintedanib (1 nM to 10 µM). ND, not detected.