Journal: Nature Communications
Article Title: NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells
Figure Lengend Snippet: Increased NOTCH activation facilitates EHT. a Schematic diagram of experiments. D4 HE cultured in presence of DAPT for 4 days (D4 + 4) or 1 day (D4 + 1), or DMSO (control). CD144 + endothelial and CD43 + blood cells were analyzed following 4 days of culture. b Flow cytometric analysis demonstrates that NOTCH activation facilitates EHT as evidenced by the decrease in hematopoietic activity when DAPT is added only from D4 to D4 + 1. Representative contour plots from three independent experiments are shown. c Frequencies of endothelial and blood cells in HE cultures treated with DAPT or DMSO (control). Results are mean ± s.e.m. for at least three independent experiments; two-way ANOVA Bonferroni post-hoc test, * p
Article Snippet: Purified CD31+ HE were then plated at a density of 20,000 to 30,000 cells/cm2 on collagen IV-coated plates (1 μg/cm2 ) that were either co-coated with IgG-Fc fragments or human DLL1-Fc (made in-house), in IF9S media supplemented with FGF2, VEGF, EGF, IGF-I, IGF-II, TPO, IL-6 (50 ng ml−1 ), SCF (20 ng ml−1 ), IL-3, FLT3L (10 ng ml−1 , Peprotech), and ROCK inhibitor (5 μM, Cayman Chemicals), and where specified, DMSO (1:1000, Fisher Scientific) or DAPT (10 μM, Cayman Chemicals), and cultured in normoxia (20% O2 , 5% CO2 ).
Techniques: Activation Assay, Cell Culture, Flow Cytometry, Activity Assay