Detection of HSPCs and myeloblasts near bone trabeculae, vasculature, and megakaryocytes. (A) Permutation testing approach: observed locations of cell types are randomized ∼5000 times per each case, and then proportion of cell type(s) within region proximal to bone (25 μm), vasculature (25 μm), or megakaryocytes (50 μm) is compared to proportion in randomized location (supplemental Figures 8-10). (B) (Top) Representative MxIF images from a young marrow (WCM68), including whole tissue image (left). Representative juxtabecular area contains HSPCs (arrowhead: CD34⁺CD38^+/−CD117^–) and myeloblasts (arrows: CD34⁺CD38^+/−CD117⁺). (Bottom) Representative MxIF images from a young marrow (WCM66), including whole tissue image (left). Representative focus surrounding a CD34⁺ endothelium-lined arteriole contains HSPCs (arrowhead: CD34⁺CD38^+/−CD117^–) and myeloblasts (arrows: CD34⁺CD38^+/−CD117⁺). (C) HSPCs are located similarly close to bone trabeculae and vasculature for all age groups (y-axis = HSPCs as proportion of all nucleated cells; x-axis = distance bins from nearest bone trabeculae or vessel concatenated from all age groups). (D) Representative MxIF images from a young marrow (WCM60), including whole tissue image (left). Representative focus around a CD61⁺ megakaryocyte shows nearby HSPCs (arrowhead: CD34⁺CD38^+/−CD117^–) and a myeloblast in the vicinity (arrows: CD34⁺CD38^+/−CD117⁺). (E) The absolute number of HSPCs within 50 μm of CD61+ megakaryocyte borders was greater in ≤20 year marrows than in >60 year marrows (118.14 ± 53.54 vs. 48.91 ± 19.28; P < .0001), and greater in 20-60 year marrows than in >60 marrows (96.61 ± 23.02 vs 48.91 ± 19.28; P < .001).