Figure 4.
Quantitative Microscopy of Bone Organs Reveals the Reconstitution of a Human Mesenchymal Niche Including Rare Mesenchymal-Hematopoietic Physical Interactions
(A) Multidimensional confocal immunofluorescence imaging allows for the reconstitution of hOss for 3D quantitative information retrieval (left). Top view of a transverse hOss section (right) illustrating the internal bone marrow cavity (DAPI) and intense peripheral vascularization (lamimin). Scale bars, 400 μm.
(B) Top: implanted hMSCs (VENUS positive) demonstrate fate plasticity by acquiring multiple niche cell phenotypes. Scale bars, 20 μm. Bottom: fate quantification of VENUS hMSCs based on the segmentation of immunofluorescence data. Stromal cells, venus+/CD90+/stroma localization; osteocytes, venus+/ALP-/localization in bone nodules; osteoblasts, venus+/ALP+/localization at the bone and bone marrow interface; vascular associated, venus+/distance to lamimin+ vessels < 1 μm; ALP, alkaline phosphatase. n ≥ 3 biological replicates.
(C) The SDF1α protein was expressed by blood cells and also found more abundantly in VENUS cells from the VENUS-SDF1α ossicles. Right and b are magnification panels.
(D) Deep confocal analysis of hOss allows for the identification and localization of a rare HSPC subset (arrow, hCD45/CD34/CD90), found directly in contact with hMSC-derived niche cells. n = 10 events detected in 5.9 mm3 of tissue scanned. Data are represented as mean ± SEM.