HSC^α-cats occupy different BM locations composed of discrete cell types. (A-D) Quantification of HSC^α-cat localization in relation to combinatorial BM niches. Scatterplot showing 2-dimensional [2D]) distance quantification of single HSCs in relation to the entire vasculature and MKs (A) and the corresponding high-magnification images (B) or entire vasculature and bone (C). Most HSC^α-cats located proximal to the vasculature (A; Q2 and Q1) and did not simultaneously associate with MKs (A; Q2 vs Q1); almost all HSC^α-cats found close to MKs (A; Q1 and Q4) were also close to the vasculature (A; Q1 vs Q4). Dots represent 261 individual HSCs (orange; n = 6) and 584 RDs (gray; n = 6). Data points highlighted with blue circles are shown in panel B. To highlight the cells of interest in panel B (cKit⁺GFP⁺ cell), cKit and GFP signal outside the cells were computationally masked to appear black (mcKit/mGFP). (D) Scatterplot showing 2D distance quantification of single HSCs in relation to sinusoids and Cxcl12 stroma (D; 251 HSCs and 440 RDs, n = 5). (E) Graphic depiction of HSC localization in relation to dual and triple niches and quantification of their frequency in full-bone sections (n = 3). Statistical significance for panels A and C-D was assessed by 2-tailed nonparametric Mann-Whitney U test. *P < .05. Scale bars,10 μm (B). Cxcl12, Cxcl12 stroma.