Engraftment and multilineage differentiation of transplanted hBMSCs in infarcted myocardium. (A–D) Engraftment of DiI-labeled hBMSCs and TBMCs into infarcted myocardium. Numerous hBMSCs (red fluorescence) (A) are engrafted into the infarct and peri-infarct region of myocardium at 4 weeks after transplantation. In contrast, considerably fewer TBMCs (red fluorescence) are observed, mostly within the infarct area (C). B and D are the Hoffman images of A and C, respectively, showing the localization of engrafted cells. (E and F) Immunophenotypic characterization of hBMSCs that have differentiated into CMCs. Myocardial samples 4 weeks after transplantation were stained for cTnI (E) and ANP (F) (each detected with FITC-labeled secondary Ab). Transplanted DiI-hBMSCs expressed both markers and were indistinguishable from host CMCs. (G and H) Myocardial sections stained with ILB4, an EC marker, demonstrate that DiI-hBMSCs are colocalized with vascular ECs in both the infarct (G) and the peri-infarct (H) area (arrows). (I) Myocardial sections stained with α-SMA illustrate DiI-hBMSC colocalized with vascular SMCs (arrows). (J–L) FISH on hBMSC-transplanted hearts. FISH with α-sarcomeric actinin staining (J) demonstrates that transplanted hBMSCs shown in FISH-positive red fluorescence express a CMC phenotype (green fluorescence). FISH with ILB4 staining (K) demonstrates that transplanted hBMSCs shown in FISH-positive red fluorescence exhibit a vascular EC phenotype (green fluorescence). FISH with α-SMA staining (L) reveals that transplanted hBMSCs shown in FISH-positive red fluorescence express an SMC phenotype (green fluorescence). White arrows indicate FISH-positive cells stained with CMC, EC, or SMC markers; yellow arrows indicate FISH-positive cells not stained with CMC, EC, or SMC markers. Scale bars in A–I: 100 μm; scale bars in J–L: 50 μm.