HSCT and permanent changes in the MSPCs. (A) Experimental design for HSCT into lethally irradiated 3-month-old wild-type mice (time point at the end of experiment: 13 months, 13R). Age-matched control group (13A) without treatment (HSCT). Analysis of the BM niche in 13-month-old mice (13A and 13R), and Y control group (3 months). Fluorescence-activated cell sorting (FACS) analysis of isolated stromal cells with subsequent cultivation of the MSPCs. (B) Graph shows the total cell number of 4 long BM flushed bones after collagenase digestion. (C) Relative numbers of immature MSPCs (CD45/Ter119/CD31⁻ Sca-1⁺ Alcam^−/low, left) and OBCs (CD45/Ter1197CD31⁻ Sca-1⁻ Alcam⁺, right); FACS gating strategy in Landspersky et al.¹¹ Representative contour plots of collagenase-digested bones (below). (D) Number of colony-forming mesenchymal stem cells (CFU-F) of 300-plated cultured MSPCs (p4). (E) Graphs show foci/cell (left) and colocalization pixel of yH2A.X and 53BP1 in the nucleus of cultured MSPCs (p4; right). Representative IF images of yH2A.X (green) and 53BP1 (red) in compact bone-derived MSPCs (p4; below) counterstained with DAPI (4′,6-diamidino-2-phenylindole). (F) Average proportion of bluish β-galactosidase (β-gal)–stained compact bone–derived MSPCs (p4, left). White bar indicates cells with no detectable staining whereas light blue and dark blue refer to partially and strongly β-gal–stained cells, respectively; Y (n = 5), 13A (n = 6), and 13R (n = 4). The Kruskal-Wallis test was applied here between the 3 groups examined for each of the 3 β-gal staining concentrations (strong, medium, and no). Representative pictures of β-gal–stained MSPCs (right). The analysis represents 2-3 independent experiments. Scale bars, 5μm (E) and 20μm (F). ∗P < .05 (Kruskal-Wallis test: panels B-F). Data are represented as mean ± SD. Ctrl, control.