CFSE-labeled HPCs (Lin⁻, Sca-1⁻, c-Kit⁺; L−S⁻K⁺) or eHPCs (Lin⁻, Sca-1⁺, c-Kit⁺; L−S⁺K⁺) were transplanted in recipient animals. The tibia of recipient animals was analyzed between 16 and 40 hours posttransplant by intravital time-lapse 2-photon microscopy to monitor CFSE-labeled green (A) L⁻S⁻K⁺ cells and (B) L⁻S⁺K⁺ cells close to the endosteal region (brown signal, autofluorescence) of the bone. (C) Example of cell protrusions extending from transplanted L⁻S⁻K⁺ cells into the endosteum (green = CFSE, brown/red = bone surface [autofluorescence]). (D) 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 = 169 changes for L⁻S⁻K⁺ cells and n = 92 changes for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population; see also as example supplemental Video 3 for L⁻S⁻K⁺ cells and Video S4 for L⁻S⁺K⁺ cells). (E) Average variance in the change in cell volume in a 1-minute time interval of L⁻S⁻K⁺ and L⁻S⁺K⁺ cells (n = 169 changes for L⁻S⁻K⁺ and n = 92 changes for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population). (F) Average variance in the change of cell surface area in a 1-minute time interval of L⁻S⁻K⁺ and L⁻S⁺K⁺ cells (n = 169 changes for L⁻S⁻K⁺ and n = 92 changes for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population). (G) Distance of L⁻S⁻K⁺ cells and L⁻S⁺K⁺ cells to the endosteum; each dot represents a single cell; black horizontal bars depict the average distance; and red/blue horizontal bars depict the standard deviation (n = 93 for L⁻S⁻K⁺ and n = 44 for L⁻S⁺K⁺, based on at least 3 independent experiments for each cell population). (H) Percentage of HPCs (CAFC day 7-21 cells) and HSCs (CAFC days 28 and 35 cells) that adhere in 3 hours to FBMD-1 stroma cells in vitro, determined by the CAFC adhesion assay. n = at least 6 experiments for each time point. (I) Relative level of expression of CD49d and CD49e on HPCs and HSCs as determined by flow cytometry. Figure is representative of 6 independent experiments. *P < .05.