Extended Data Figure 4. Endothelial to haematopoietic conversion capture by live microscopy.

(See also Supplementary Video 1.) a, Schema detailing the experimental setting for live confocal image capture. Adult lung endothelial cells were isolated from Runx1-IRES-GFP. Then, Runx1-IRES-GFP adult lung endothelial cells were transduced with Fgrs and co-cultured with VN-ECs (HUVEC-E4ORF1). VN-ECs were differentiated from Fgrs-transduced Runx1-IRES-GFP adult lung endothelial cells by anti-human CD31 live staining (red). Live confocal images were acquired every 45 min for the duration of the experiment (see also Supplementary Video 1). b, Representative flow cytometry plots of day 10 (d10) showing that a subset of VEcad⁺CD45⁻ and VEcad⁺CD45⁺ cells also co-express Runx1-GFP. c, Single time points from live confocal image capture. Upon doxycycline-dependent conditional expression of Fgrs, flat spindle-shaped adult mouse endothelial cells rapidly transition from RUNX1⁻ to round haematopoietic-like RUNX1⁺ cells (day 0–8, white arrow). This induction phase is characterized by the transitioning endothelial cells towards haematopoietic fate. From day 8 to 20 of specification phase, RUNX1⁺ cells further moved towards a haematopoietic identity by assuming a prototypical fully formed round shape (white arrow). Following the emergence of this definite haematopoietic program, a phase of robust expansion on the vascular niche layers (VN-ECs) of these RUNX1⁺ committed converted cells from day 20 to 28 (expansion phase).