(A) Hematoxilin and eosin staining of a teratoma within the infarcted area. Note the presence of early stage hyaline cartilage (right, top) and gastrointestinal-like columnar epithelium (right, bottom). (bars: left 500 µm, middle 200 µm, right 100 µm). (B) Immunostaining for the pluripotent markers Tra-1-60 (top) and Tra-1-81 (bottom). Note the positive staining of undifferentiated human embryonic stem cell (hESC) colonies (left) and the absence of staining in cells isolated from the contracting embryoid bodies (EBs) (right) (bar: 100 µm). (C) Troponin I immunostaining of cells isolated from the contracting EBs (bar: 60 µm).

(A) The transplanted area was localized by coinjection of 2-µm fluorescent beads (green), and the grafted cells were identified by immunostaining with antihuman mitochondria antibodies (red) (bar: 50 µm). (B) (Left) Hematoxilin and eosin staining depicting a cluster of grafted human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (arrows). (Right) Immunostaining of the boxed area. In this example, enhanced green fluorescent protein-expressing hESC-CMs were used and were identified as yellow cells containing both troponin I (red) and enhanced green fluorescent protein (green) immunosignals. HC = human cardiomyocytes; R = rat (bar: 100 µm). (C) Immunostaining with antisarcomeric -actinin (red) and antienhanced green fluorescent protein (green, right) antibodies. At this stage (36 h), the grafted hESC-CMs (arrows) displayed an immature, striated pattern (bar: 12 µm). (D) Assessment of the proliferation capacity of the hESC-CMs (36 h post-grafting) using antihuman Ki-67 (green) and antitroponin I (red) antibodies (bar: 10 µm). (E) (Left) Immunostaining of the grafted area (30 days post-transplantation) using antisarcomeric -actinin (red) and anti-Cx43 (white) antibodies. (Right) Superposition of the immunostaining results with antienhanced green fluorescent protein (green) antibodies. Note the relatively organized sarcomeric pattern (bar: 20 µm). (F) Immunostaining of the transplanted fluorescently labeled (yellow) hESC-CMs with anti-Ryanodine antibodies. Nuclei are counterstained with To-Pro3 (blue) in all immunofluorescent images.

(A) Identification of the grafted hESC-CMs at the scar’s center using antienhanced green fluorescent protein (green, left) and antisarcomeric -actinin (red, middle) antibodies. (Right) Superposition of both images. The scar was identified using anticollagen antibodies (blue) (bar: 80 µm). (B) Immunostainings of the transplanted hESC-CMs at the infarct border zone using antienhanced green fluorescent protein (green) and antitroponin I (red, right) antibodies (bar: 75 µm). (C) Identification of the grafted hESC-CMs with antihuman-human leukocyte antigen antibodies. (Top) Immunohistochemistry results (bar: 100 µm). (Bottom) Immunofluorescent staining using antihuman-human leukocyte antigen (green) and antitroponin I (red) antibodies (bar: 75 µm). (D) Development of gap junctions (Cx43 immunostaining, white) between the grafted cells (prelabeled with Vybrant-CFDA) (green, left) and host cardiomyocytes (arrows). Cardiomyocytes were identified using antitroponin I antibodies (red, middle) (bar: 60 µm). Abbreviations as in Figure 2.

(A) Polymerase chain reaction-based deoxyribonucleic acid amplification using human-specific primers at various time points after transplantation. (B) Coimmunostaining with anti-LacZ (red) and antitroponin I (green) antibodies (bar: 100 µm). (C) The nLacZ-expressing cells (blue) were isolated using laser microdissection (shown before [left] and after [right] laser microdissection) (bar: 200 µm). (D) Myosin light chain-2a expression in the microdissected nLacZ-expressing cells, in a remote left ventricular (LV) site, and in a saline-injected heart. Contracting embryoid bodies served as positive controls. d = days; hESC-CMs = human embryonic stem cell-derived cardiomyocytes.

(Top and middle) M-mode echocardiographic images demonstrating post-infarction remodeling with left ventricular dilatation and functional deterioration in the saline- and nonmyocyte transplantation groups, respectively. (Bottom) Similar images in the human embryonic stem cell (ESC)-derived cardiomyocyte grafting group. Note the absence of significant left ventricular dilatation (bar: 0.5 cm).

(A) Changes in fractional shortening in individual animals before (post-injury baseline) and 30 and 60 days after cell grafting. (B to D) Changes in the average fractional shortening (B), wall motion score index (C), and left ventricular end-diastolic diameter (D) values in the human embryonic stem cell (ESC)-derived cardiomyocytes (green), nonmyocyte (purple), and saline-injection (red) groups. (E) Comparison of the lung weight to tibia length ratio between the groups. The p values were Bonferroni-adjusted for 3 comparisons: *p < 0.05; p < 0.01; p < 0.005. MI = myocardial infarction.