Fig 2: ROS scavengers rescued the EndMT induced by PSC-Fe2O3. a Intracellular ROS of PSC-Fe2O3 treated HUVECs in the absence and presence of mannitol or AA. b The level of VE-cad, CD31, α-SMA and FSP determined by western blot. c Expression of VE-cad (green), CD31 (green), α-SMA (red) and FSP (red) for HUVECs by confocal microscopy. Data are mean ± SD, n = 3, one-way ANOVA with Fisher’s least significant difference test (LSD-t), ** p < 0.01, significant compared to control

Fig 3: Characterization of the occurrence of the EHT during the hematopoietic specification. A Morphological features of adherent cells and suspension cells climbed out from aforementioned adherent cells at the 11th day after the hematopoietic differentiation from hESCs (10× magnification, scale bars 100 μm, far left one photo; 20× magnification, scale bars 50 μm, right three photographs). B CLSM images of the immunostaining for VE-Cadherin and CD45 in the differentiated cells at day 11 undergoing the EHT. The colors of green, red and blue represent the staining for CD45, VE-Cadherin and nuclei, respectively. (Original magnification, 20×, scale bar 40 μm, upper panel; 40×, scale bar 50 μm, lower panel.). C Flow analysis for HEPs (CD31+CD45+ cells or CD144+CD45+ cells) at the 11th day during the hematopoiesis. D The dynamic gene expression of the indicated HSC-specific transcription factors by RT-qPCR during the hematopoiesis. E The transduced efficiency of lentivirus containing EGFP and mCherry reporters in hESCs was evaluated by fluorescent microscopy. (Original magnification, 10×, scale bar 100 μm). F The aggregate-forming ability of engineered hESCs seeded on the low adhesion plate was not affected by the transduction with the lentivirus. (Original magnification, 4×, scale bar 10 μm) G The generation of EGFP and mCherry double-positive hematopoietic cells during the development of the hematopoiesis was visualized by living cell fluorescence trace system. (Original magnification, 4×, scale bar 100 μm). Data represent the mean ± SEM. *p < 0.05 and ns (no significance)

Fig 4: Gene expression (mRNA) of endothelial markers A, PECAM1, B, CDH5 and C, NOS3; mesenchymal markers D, TAGLN and E, CNN1; and collagens F, COL1A1 and G, COL3A1 by semi‐quantitative RT‐qPCR of HUVEC under stimulation with IL‐1β or co‐stimulation with IL‐1β/TGF‐β2, both in ECMed and in ASC‐CMed, for five days. Data were analysed by one‐way ANOVA with Sidak's multiple comparison test for the groups ECMed/IL‐1β/TGF‐β2 vs ASC‐CMed/IL‐1β/TGF‐β2; P‐values for the Sidak's multiple comparison test are shown in the figure. Values represent mean ± SEM of four independent experiments in duplicate

Fig 5: Hypoxia enhanced the hematopoietic differentiation by promoting the EHT progression. A Flow cytometry analysis for HEPs (hESC-derived CD144+CD34+ cells) at day 11 after the hematopoietic differentiation under the hypoxia or normoxia conditions. B CLSM images of the immunostaining for VE-Cadherin and RUNX1 at day 15 cells during the hematopoietic specification under the normoxia or hypoxia conditions. The colors of green, red and blue represent the staining for RUNX1, VE-Cadherin and nuclei. (Original magnification, 40×, scale bar 50 μm). C Relative endothelial gene expression of differentiated cells at different time points under the normoxia or hypoxia conditions. D Flow cytometry analysis for HPCs (hESC-derived CD34+CD45+ cells) during the hematopoiesis under the hypoxia or normoxia conditions. E T lineage potential of HPCs at day 15 after the hematopoietic differentiation following 4 weeks of culture for induction of T cells. F and G Cell cycle distribution was determined by flow cytometry after the hematopoietic differentiation under the normoxia or hypoxia conditions. The percentage of S phase was significantly increased in hESC-derived CD34+CD45+ cells under the normoxia conditions. H CLSM images of the immunostaining for ROS1 in hESC-derived CD34+CD45+ cells under the normoxia or hypoxia conditions. The colors of red and blue represent the staining for ROS1 and nuclei. (Original magnification, 40×, scale bar 50 μm). I Gene expression analysis of cell cycle gene (P27) and apoptosis gene (BAX) by RT-qPCR at different time points during the hematopoietic differentiation under the normoxia or hypoxia conditions (n = 3, mean ± SEM). J Gene set enrichment analysis for the identified genes involved in HIF-1a induced hypoxia during the hematopoietic specification. K RT-qPCR analysis for the expressions of HIF-1α, VEGFA, NOTCH1 and RUNX1. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ns (no significance)