Discrete patterns of homing of leukemic and pre-LSCs compared with normal stem and progenitor cells. (A) In vivo imaging of HSCs, GMPs, pre-LSCs (MLL-AF9 transduced GMPs, expanded in vitro), and LSCs (MLL-AF9 transduced GMPs, in vivo expanded, from leukemic mice) homing to the bone marrow cavity of lethally irradiated osteoblast reporter recipient mice. Red with white outline represents injected HSCs, GMPs, pre-LSCs, or LSCs stained ex vivo with Vybrant DiD dye; green, osteoblasts; and blue, bone matrix. White bar represents 10 μm. The shortest, 3-dimensional distance from each HSC, GMP, pre-LSC, or LSC to the closest osteoblast was measured for each cell. (B) Pre-LSCs (MLL-AF9 GMPs expanded in vitro) homed 8.7 μm (median; range, 0.5-42 μm) from osteoblasts compared with LSCs that homed 23.7 μm (median; range, 0-79.2 μm) from osteoblasts (P < .01). Individual data point represents an individual cell. n = 3 or 4 biologic replicates. (C) HSCs homed 12.8 μm (median; range 0-42.1 μm) from osteoblasts compared with GMPs that homed 21.0 μm (median; range, 1.0-89.2 μm) from osteoblasts (P = .03). n = 3 biologic replicates per condition. (D) LSCs (MLL-AF9–transduced GMPs, expanded in vivo from leukemic mice) proliferate and form clusters in vivo. LSCs were stained with either Vybrant DiD (Invitrogen, red) or DiI (Invitrogen, white) and mixed in equal proportions before injection into lethally irradiated recipient mice. After 48 hours, clusters were observed. In all cases, clusters were made of a single color (red or blue), confirming clonal LSC expansion in vivo. All images were acquired using a 25× water immersion objective (NA 0.9) on a custom-made confocal/two-photon microscope and acquisition software (1). Image analysis was performed with ImageJ and measurements were obtained using Photoshop CS4 extender.