Fig. 4

Cell cycle progression is necessary for EHT. a (Left panel) tSNE plot of the merged data from EHT D3 and EHT D5. Cells were coloured based on the cell cycle phase inferred from scRNA-seq data. (Middle panel) Cell cycle phase along differentiation trajectory, from endothelial cells (right) to haematopoietic and mesenchymal branches (left). (Right panel) Pie charts showing the percentage of cells in G1, G2/M, and S phase in each of the clusters. b EdU staining and flow cytometry were used to monitor the cell cycle profile of CD43− and CD43+ cells during differentiation. Results are representative of 3 independent experiments. c–e At EHT D3, media were supplemented either with 0.1% DMSO (CTRL) or with 0.1 μg/ml Nocodazole (Noc). After 48 h, at EHT D5, cells were either c processed for EdU staining and flow cytometry to monitor their cell cycle profile, d washed to remove Nocodazole and further cultured in a CFU assay to reveal their differentiation potential, or e stained for flow cytometry to monitor the distinct populations previously identified (P1, mesenchymal cells; P4, endothelial cells; P2 and P5, erythroid progenitors; P3 and P6, HPCs). Results are representative of 3 independent experiments. Data in d and bar plot in e are mean ± SEM of 3 independent experiments, with statistical significance compared to control as *P < 0.5, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test, and representative of 2 distinct hPSC lines. Degrees of freedom = 4; t values for the distinct colonies in d are BFU-E = 39.62, CFU-E = 15.74, CFU-GM = 6.871, CFU-G = 4.271, CFU-M = 4.867, and CFU-GEMM = 5.196; t value in e = 15.05