Figure 4 - Human MSC homing to mouse bone marrow.

(a) Images obtained from intravital confocal microscopy of the parasagittal region of the calvarium in representative NOD/SCID mice at 1 h after infusion of the indicated cells (top). Results shown in the lower panels are representative images of one mouse at 1 h (left) and 24 h (right) after infusion of HCELL⁺ MSCs. Scale bar, 250 m. Bar graph represents the number of MSCs localized to the bone marrow 1 h after infusion, mean s.e.m. for n = 5 experiments. P < 0.001 for comparison of HCELL⁺ MSCs to each other group. The middle bar in the graph ("MSCs") combines data from untreated MSCs and BT-MSCs, for which the results were identical. (b,c) In vivo confocal and two-photon microscopy showing extravasation of human HCELL⁺ MSCs in mouse marrow and subsequent migration to the endosteal surface after adoptive transfer; images are representative of n = 12 experiments. (b) DiD-labeled MSCs (red) primarily line the vessel walls (visualized by Angiosense 750, green) 1 h after transfer (left); at 24 h (right), MSCs have extravasated. The two images were obtained at the same site with the identical tissue plane. White boxes correspond to higher-magnification images. Most MSCs are intravascular at 1 h (arrows), but by 24 h, cells have infiltrated the marrow parenchyma (arrows). (c) Second harmonic–generation imaging microscopy reveals close juxtaposition between DiD-labeled cells (red) and bone (collagen, blue) after 9 d. Bone image at right is 10 m above the image at left, showing the close proximity of the endosteum to the cells (arrows). Scale bar, 100 m. (d) Immunofluorescence images of frozen calvarium sections from NOD/SCID mice that received intravenous injections of HCELL⁺ MSCs or BT-MSCs as indicated (5 10⁶ cells per mouse). The leftmost image in each row is a phase light microscopy image of the bone section. Twelve weeks after transplantation, the mice were killed and their skulls isolated, snap frozen and sectioned. Images are representative of n = 3 separate experiments (with duplicate mice in each group for each experiment). Locations of bone and of bone marrow are designated by B and M, respectively; scale bars, 50 m. Sections were stained for human CD44 (green) and human osteocalcin (red); in the merged image, arrows indicate cells staining for CD44 in the vicinity of osteocalcin deposits (yellow). Specificity of mAb to human osteocalcin was confirmed by staining MSC before and after osteogenic differentiation in vitro (see Supplementary Fig. 6 online).