Fig 2: Iron deficiency does not block endothelial to haematopoietic transition. (a) A schematic timeline of an experiment in blast culture. Flk1+ cells were plated in blast mix on day 0, treated 24 hours later and profiled by flow cytometry for expression of endothelial and hematopoietic markers after another 24 hours. The treatments were: nothing (control), DFO 50 µM (iron-deficient conditions), DFO + ferric ammonium citrate 200 µM (simultaneous neutralization of iron deficiency, or iron compensation), Fe 200 µM (ferric ammonium citrate for iron excess conditions). (b) A flow cytometry plot showing expression of CD41 against VE-Cad in day 2 blast (24 hours of treatment). According to the expression of these markers, each blast culture can be divided into 4 quadrants: VE-Cad- CD41- = vascular smooth muscle cells (VSM); VE-Cad+ CD41- = endothelial cells (EC); VE-Cad+ CD41+ = pre-hematopoietic progenitor cells (Pre-HPC); VE-Cad- CD41+ = hematopoietic progenitor cells (HPC). The experimental conditions are shown from left to right. The Tukey’s boxplots of the different cell type frequencies from flow cytometry experiments are shown for HPC (c), Pre-HPC (d), VSM (e) and EC (f). Box whiskers show minimum and maximum, the line inside boxes shows median. For control and DFO groups n = 10; for DFO + Fe and Fe groups n = 6. All groups were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. For (c) #p = 0.049 and *p = 0.027, for (e) *p = 0.032, for (d,f) N.S. = not significant.

Fig 3: KDM4A and KDM4C Mediate mESC Differentiation to Endothelial Cells(A) Diagram showing experimental protocol for siRNA treatment. mESC were transfected on days 1 and 3 to achieve maximum knockdown of protein. The cells were differentiated in medium containing BMP-4, bFGF, and VEGF for 6 days.(B) The effect of Kdm4a and Kdm4c knockdown on expression of endothelial-specific markers. Efficiency of Kdm4a and Kdm4c knockdown was 60%–80%. Oct4 expression was not suppressed by either Kdm4a or Kdm4c knockdown (n = 4 per group). Both Kdm4a and Kdm4c knockdown resulted in 90% and 80%, respectively, reduction in Flk1 and VE-cadherin expression on day 6 (n = 3 independent experiments).(C) Flow cytometry assessment of cell surface expression of Flk1 and VE-cadherin on day 6 of differentiation following treatment with scrambled, Kdm4a, Kdm4c, or Kdm4a and Kdm4c siRNAs. Quantification in the bar graph shows that the percentage of FLK1 and VE-cadherin double positive cells was reduced from 17% to less than 5%; FACS profile is representative (n = 3 independent experiments). Quantification values are mean ± SD; p < 0.05. Combining Kdm4a with Kdm4c siRNAs did not further suppress differentiation.(D) Treatment of mESCs with BMP-4 alone (2 ng/ml) is sufficient to induced Kdm4a expression at 24-hr post-stimulation. Treatment with bFGF alone (10 ng/ml) induced Kdm4c expression at 48-hr post-stimulation. Error bars represent mean ± SD (n = 3 independent experiments). VEGF stimulation did not induce either KDM4A or KDM4C expression during the 48-hr period.(E) Kdm4a and Kdm4c overexpression substitutes for BMP-4 and bFGF. mESCs were transfected with Kdm4a and Kdm4c and cultured in medium containing only VEGF. FACS analysis on day 6 demonstrated that the fraction of double-positive cells was 20%. Bar graph summarizes data from (n = 3) three independent experiments.

Fig 4: Expression of KDM4A and KDM4C following mESC Differentiation into Endothelial Cells(A) Flow sorting diagram of mESCs differentiated to endothelial cells stained with anti-FLK1 and anti-VE-cadherin antibodies. Cells were separated into FLK1/VE-cadherin-double positive endothelial cells or double-negative control cells.(B) Time course of gene expression assessed by qRT-PCR for the pluripotency genes Oct4 and Nanog as well as for endothelial genes Flk1 and VE-cadherin during the 6-day differentiation period (D1 to D6; ES are mESCs); error bars represent mean ± SD (n = 3 independent experiments, p < 0.05 by ANOVA).(C) qRT-PCR screen for 28 known histone demethylases was carried out in mESCs differentiated into FLK1/VE-cadherin-double positive endothelial cells (D6), double-negative control cells (D6), and adult primary mouse endothelial cells. Expression of KDM4a and KDM4c was markedly increased in mESC-derived and mouse endothelial cells as assessed by two-way ANOVA (all values are normalized to 18S in undifferentiated mESCs). Values are mean ± SD (n = 3 independent experiments, *p < 0.001 by two-way ANOVA).(D) Western blotting carried out using anti-KDM4A and anti-KDM4C antibodies in sorted FLK1/VE-cadherin-double-positive cells derived from mESCs at day 6 of differentiation. Representative examples from three independent experiments are shown.(E) Time course of Kdm4a and Kdm4c expression determined by qRT-PCR during the day 1 to day 6 period of endothelial cell differentiation from mESCs. Values are mean ± SD (n = 3 independent experiments); gene expression in undifferentiated cells (ES) is set as 1; p < 0.05 by ANOVA.

Fig 5: KDM4A and KDM4C Bind to Histones Associated with Flk1 and VE-cadherin Promoters, Respectively(A) Differentiated cells were subjected ChIP-qPCR analysis using control IgG, anti-KDM4A, and anti-KDM4C antibodies (see Experimental Procedures). The ChIPs were subjected to qPCR using oligonucleotides to amplify the promoter regions of VE-cadherin, Flk1, and the transcription factor Er71. Chromatin binding to IgG-ChIP was considered background. Flk1 promoter was enriched in KDM4A ChIP, whereas VE-cadherin promoter was enriched in KDM4C ChIP. Er71-promoter was not detectable (ND) in either ChIP. Values are mean ± SD (n = 3 independent experiments).(B) Kdm4a knockdown prevented enrichment of Flk1-promoter in the ChIP assay, whereas Kdm4c knockdown prevented enrichment of the VE-cadherin promoter. Values are mean ± SD (n = 3 independent experiments).(C) ChIP assay was performed using an anti-histone H3 tri-methylation at lysine (K9) antibody to assess methylation of histones at the promoters of VE-cadherin, Flk1, and Er71 in undifferentiated mESCs; values are mean ± SD (n = 3 independent experiments).(D) Histone 3 tri-methylation at lysine 9 (H3K9) was assessed by western blotting in mESCs and FLK1/VE-cadherin-double-positive cells. An antibody against total histone H3 was used as loading control. The blot is representative of three independent experiments. Bar graph (mean ± SD) shows significant reduction of histone H3 tri-methylation (K9) in endothelial cells as compared to undifferentiated ESCs (p < 0.05 by Student’s t test).