HSPCs polarize upon specific heterotypic interactions. (A) Schematic description of the experimental system to evaluate interactions between different stromal or nonstromal cell types of interest (listed in blue) and different hematopoietic cell types isolated at different stages of differentiation (listed in black). (B) As schematized, the HSPC polarization index (Pi) was defined as the ratio between the distance from the point of contact to the centrosome (d) and the HSPC length (D). Representative images of HSPCs with a Pi close to 0 (left) and or close to 1 (right). Actin filaments appear in red, microtubules in green, the centrosome in white, and DNA in blue. Arrowheads highlight the point of contact. Scale bar = 5 µm. (C) SuperPlot of the polarization index of HSPCs in contact with osteoblasts, endothelial cells, and skin fibroblasts. Each color represents a biological replicate (three replicates; osteoblasts hFOB n_total = 135, endothelial cells HUVEC, n_total = 178, and skin fibroblasts BJ, n_total = 149). For each replicate, the median appears as a large diamond, triangle, or hexagon with the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (D) SuperPlot of polarization index of HSPCs in contact with mouse liver–derived stromal cell lines that either support HSPC regeneration capacities (AFT024) or not (BFC012). Each color represents a biological replicate (three replicates; AFT, n_total = 99; BFC, n_total = 106). For each replicate, the median appears as a large diamond or triangle of the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (E) SuperPlot of polarization index of HSCs (CD38⁻, n_total = 97), CMPs (CD38⁺/CD33⁺, n_total = 140), primary human monocytes (CD14⁺, n_total =85), nonactivated (CD3⁺, n_total = 115) or activated (CD3⁺, n_total = 138) T cells, and immortalized T lymphocytes (Jurkat cells, n_total = 82) in contact with osteoblasts. Each color represents a biological replicate (three replicates). For each replicate, the median appears as a large hexagon, diamond, triangle, disk, square, or inverted triangle of the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (F) Representative images of HSPC/CD34⁺ (upper left), monocyte (lower left), inactivated T cell (upper right,) and activated T cell (lower right) interacting with osteoblasts. Actin appears in red, centrosome in white, and DNA in blue. Arrowheads highlight the point of contact. Scale bar = 10 µm.