HSCs are found in clusters in bone marrow in aged animals.

(A, B) Representative 3D confocal pictures of a whole-mount femur from a young (panel A) and an aged (panel B) mouse. HSCs are indicated by yellow arrowheads. Dashed lines in panel B highlight clustered HSCs. Lineage markers CD41 and CD48 are shown in white; CD150 in red and endothelial cells (IV injected VE-Cadherin CD144 and PECAM-1 CD31) are in magenta. (C) Distance between HSCs in young and aged femurs. (D) Percentage of HSCs located in 20 μm intervals from the closest HSC in young and aged femurs. (E) Pie charts depicting the percentage of HSCs found in clusters (2 cells or more adjacent to each other) or as single cell in young and aged mice. Imaging data refer to 27 young and 14 aged longitudinal shaved whole-mount cross-sections from 2 young and 2 aged mice; 144 young HSCs and 394 aged HSCs. (F, G) Representative 3D confocal pictures of whole-mount femur analyses from young recipient mice transplanted with young (panel A) and aged (panel B) RFP+ HSCs. Donor-derived RFP+ HSCs are marked by yellow arrowheads. Lineage markers CD41 and CD48 are shown in white, CD150 in green and endothelial cells (IV injected VE-Cadherin CD144 and PECAM-1 CD31) are in magenta. All donor-derived cells are RFP+ (red). (H) Distance between donor-derived RFP+ young and aged HSCs in the femurs of young recipient mice. (I) Percentage of donor-derived RFP+ young and aged HSCs located in 20 μm intervals from the closest donor-derived RFP+ HSC in the femurs of recipient mice. (J) Pie charts depicting the percentage of donor-derived RFP+ young and aged HSCs found in clusters (2 cells or more adjacent to each other) or as single cell in the femurs of young recipient mice. Imaging data refer to 8 young and 4 aged longitudinal shaved whole-mount cross-sections from 2 young and 2 aged mice; 75 young HSCs and 108 aged HSCs. The primary data the figure is based on are provided in S1 Data. HSC, hematopoietic stem cell; IV, intravenous; Lin, lineage; RFP, red fluorescent protein.