Extended Data Fig. 3: Determination of the origin of the cells in the femoral grafts.

a, Experimental strategy to determine whether MSCs in the grafts are derived from the grafts. b, Left: representative flow cytometry plots of Nestin-GFP⁺ cells in CD45^–TER-119^–CD31^–CD51⁺CD140α⁺ MSCs isolated from the host femurs (top) and the grafts (bottom) in the experiment shown in a. Right: the frequency of the Nestin-GFP⁺ population within the CD45^–TER-119^–CD31^–CD51⁺CD140α⁺ fraction in the host femurs and the grafts. 6 host femurs and 6 grafts from 6 host mice. c, Experimental strategy to determine whether MSCs in the grafts are derived from the hosts. d, Left: representative flow cytometry plots of Nestin-GFP⁺ cells in CD45^–TER-119^–CD31^–CD51⁺CD140α⁺ MSCs isolated from the host femurs (top) and the grafts (bottom) in the experiment shown in c. Right: the frequency of the Nestin-GFP⁺ population within the CD45^–TER-119^–CD31^–CD51⁺CD140α⁺ fraction in the host femurs and the grafts. 6 host femurs and 6 grafts from 6 host mice. e, Experimental strategy to determine whether ECs in the grafts are derived from the grafts. f, Confocal z-stack projection of Cdh5-CreER; iTdTomato (red) graft transplanted to WT mice and stained for CD31⁺CD144⁺ (white) vasculature. Scale bar, 1000 µm; three independent experiments yielded similar results. g, Left: representative flow cytometry plots of TdTomato⁺ cells in CD45^–TER-119^–CD31⁺SCA-1^highCD62E^low AEC fraction from the host femurs (top left) and the grafts (bottom left) and in CD45^–TER-119^–CD31⁺SCA-1^lowCD62E^high SEC fraction from the host femurs (top right) and the grafts (bottom right) in the experiment shown in e. Right: the frequency of the TdTomato⁺ cells in AEC and SEC fractions in the host femurs and the grafts. 6 host femurs and 6 grafts from 6 host mice. h, Experimental strategy to determine whether ECs in the grafts are derived from the hosts. i, Confocal z-stack projection of WT graft transplanted to Cdh5-CreER; iTdTomato (red) mice and stained for CD31⁺CD144⁺ (white) vasculature. Scale bar, 1000 µm; three independent experiments yielded similar results. j, Left: representative flow cytometry plots of TdTomato⁺ cells in CD45^–TER-119^–CD31⁺SCA-1^highCD62E^low AEC fraction from the host femurs (top left) and the grafts (bottom left) and in CD45^–TER-119^–CD31⁺SCA-1^lowCD62E^high SEC fraction from the host femurs (top right) and the grafts (bottom right) in the experiment shown in h. Right: the frequency of the TdTomato⁺ cells in AEC and SEC fractions in the host femurs and the grafts. 6 host femurs and 6 grafts from 6 host mice. k, Schematic of the determination of the origin of haematopoietic cells in the grafts. l, The frequency of CD45.1⁺ and CD45.2⁺ cells in the whole BM cells, HSCs and differentiated haematopoietic cells in the host femurs and the grafts. 6 host femurs and 6 grafts from 6 host mice. Data are mean ± s.e.m. The diagrams in a, c, e, h and k were created using BioRender. Takeishi, S. (2025) https://BioRender.com/9d3nv16.

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