HSCs from aged mice (20- to 24-month-old animals) were fluorescently labeled (CFSE or CTO) and transplanted into young recipient animals. Tibiae from recipient animals were analyzed between 16 and 40 hours after transplantation. (A) Quantification of cell protrusion movement, determined as the change in volume over time (interval of 1 minute) of transplanted L⁻S⁻K⁺ and L⁻S⁺K⁺ cells, as described in supplemental Experimental Procedures (n = 92 changes for young L⁻S⁺K⁺ cells and n = 74 changes for L⁻S⁺K⁺ cells, based on at least 3 independent experiments for each cell population; see also as example supplemental Video 5 for aged L⁻S⁺K⁺ cells in addition to supplemental Video 4 for young L⁻S⁺K⁺ cells). (B) Average variance in the change in cell volume in a 1-minute time interval of L⁻S⁻K⁺ and L⁻S⁺K⁺ cells (n = 92 changes for L⁻S⁻K⁺ and n = 74 changes for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population). (C) Average variance in the change of the cell surface area in a 1-minute time interval of L⁻S⁻K⁺ and L⁻S⁺K⁺ cells (n = 92 changes for L⁻S⁻K⁺ and n = 74 changes for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population). (D) Distance of young and aged transplanted L⁻S⁺K⁺ cells to the endosteum; black horizontal bars depict the average distance (n = 44 for young L⁻S⁺K⁺ cells and n = 50 for aged L⁻S⁺K⁺ cells, based on at least 3 independent experiments for each cell population). (E) Percentage of young and aged HSCs (CAFC day 28 cells) that adhere to FBMD-1 stroma cells in vitro, determined by the CAFC adhesion assay (n = 6 experiments). (F) Cytoskeletal cell polarity in young and aged HSCs (Lin⁻, Sca-1⁺, c-Kit⁺) upon adhesion to fibronectin (determined by staining for F-actin [red] and microtubules [green]). (G) Percentage of polar aged and young HSCs upon adhesion (quantification of data from panel F; n = 3 with at least 20 cells analyzed per experiment). (H) Level of expression of CD49d and CD49e on young and aged HSCs as determined by flow cytometry. Figure is representative of 6 independent experiments. *P < .05.