Calcifications and bone formation in the lesioned heart after injection of enriched MSCs. (A) Methylene blue–stained semithin section through the infarcted area of a heart 29 days after injecting 2 × 105 enriched MSCs (passage 3). Transmission light microscopy revealed an encapsulated formation (arrows) filling out almost the entire width of the scar. Polarization microscopy showed fluorescent areas within the amorphous material indicating calcifications (panel A inset). (B) Von Kossa staining evidenced massive calcifications (black deposits) 29 days after injecting 1 × 105 MSCs (passage 6) into the cryoinjured mouse heart. (Panel B inset) Similar calcifications were seen using von Kossa staining in an LCA mouse heart 28 days after injecting 1 × 105 MSCs (passage 5). (C) Combined van Gieson/von Kossa staining (same heart as shown in panel B) demonstrated that the calcifications (black, marked by arrows) were restricted to the infarcted area (red) and (D) contained high numbers of the injected EGFP+ MSCs (green, subsequent section). (E) Immunostaining with osteocalcin (Cy3, red) demonstrated that the injected EGFP+ MSCs (green) were cemented within trabecular-like bone structures (sample obtained from same heart as shown in panel B). (F) Similar calcifications but of smaller size were revealed by von Kossa staining (arrow) 21 days after injecting only 1 × 104 enriched MSCs (passage 6). The infarcted area of the heart is characterized by the thinned-out ventricular wall. Inset shows the area of calcification at higher magnification. Bar represents 180 μm (panel A), 550 μm (panel A inset), 650 μm (panel B), 1300 μm (panel B inset), 600 μm (panels C,D,F), 50 μm (panel E), 100 μm (panel F inset). See “Materials and Methods; Image acquisition and preparation” for microscopy details.
