Engraftment and expansion of transplanted 5FP-marked cells in the BM in vivo. Lin⁻ cells transduced with 5FPs were infused into mice after total body irradiation. Bisected whole sternal mounts were examined at different time points by confocal and 2-photon microscopy (A-F). Peripheral blood nucleated cells from the same time point are shown to the right of each sternal marrow image (G-I). For all images, tiled z-stacks were collected over large volume (2.5-mm × 1.2-mm × 300-μm) of the mouse BM tissue and computationally stitched. (A-E) Different mice, each receiving the same population of Co 5FP–transduced donor cells. (F) Different experiment of Mix 5FP–transduced donor cells. Related images are found in supplemental Videos 4 and 5. (A-B) Endosteal engraftment occurring early in BM regeneration (day 4 and 8 after transplantation, respectively); clusters of cells marked in wide variety of colors were visible in close proximity to the bone edge (SHG, white). (C) By day 11, multiple cell clusters are located in close proximity to the bone and progressing centrally over time. (D) By day 19, multiple uniquely color-marked clones are distinguishable over the entire marrow fossae. (E) By day 34, one large clone (green) has expanded to occupy the entire fossae. However, the peripheral blood from the same time point (I) continues to show the presence of multiple clones contributing to hematopoiesis. (F) Day 120 sternum is shown from another experiment, with animals receiving Mix 5FP–transduced BM, similarly showing a very large single clone occupying most of the sternal marrow space. Scale bars represent 200 μm (A-F), 50 μm (G-H).