Bone formation origins from the MSC fraction of BM. (A) Cytosolic and extracellular (some marked by arrows) osteocalcin staining (Cy3, red) proved bone formation in a cryoinfarcted heart 13 days after injecting 5 × 10⁶ EGFP⁺ whole BM-derived cells (green). The osteocalcin accumulation in the injected (EGFP⁺) cells revealed that these produce the bone tissue. Von Kossa staining in the same also heart showed calcifications (panel A inset). (B) Distinct calcifications (von Kossa staining) inside the lesioned, thinned-out ventricular wall of a cryoinfarcted heart harvested 369 days after injection of 1 × 10⁶ EGFP⁺ whole BM cells. Osteocalcin staining (Cy3, red) of a subsequent section of the same heart revealed distinct bone formation without enclosed cells (panel B inset). Nuclei were stained with Hoechst dye (blue). (C) Prominent engraftment of EGFP⁺ cells (green) into the lesioned area 28 days after LCA and consecutive cytokine-induced mobilization of BM cells. Nuclei were stained with Hoechst dye (blue). CD45 staining (magenta) proved the hematopoietic origin of the engrafted cells (panel C inset). (D) Statistics of hearts with pathological abnormalities after infarction and injection of enriched MSCs (4 LCAs), whole BM cells (WBM, 2 LCAs), and controls (injection of vehicle, fibroblasts, hematopoietic progenitor cells, and mobilization of BM cells in reconstituted mice, 32 LCAs). P < .001 for MSCs vs WBM; P < .001 for MSCs vs control; and P = .002 for WBM vs control (2-sided Fisher exact test). Bar represents 24 μm (panel A), 50 μm (panel A inset), 750 μm (panel B), 380 μm (panel B inset), 940 μm (panel C), 40 μm (panel C inset). See “Materials and Methods; Image acquisition and preparation” for microscopy details.