(A) Survival rates over 6 weeks after cell transplantation in the animals injected with human muscle derived cells or control phosphate-buffered saline (PBS) (Kaplan-Meier survival curve, p = 0.13). (B) Echocardiography performed 2 and 6 weeks after cell transplantation demonstrated smaller left ventricular areas in end-diastole of hearts injected with CD56⁺CD34⁺CD144⁺ cells when compared with those in the other groups (baseline: data from NOD/SCID mice without infarction and cellular injection). (C) The CD56⁺CD34⁺CD144⁺ myoendothelial cell group also displayed greater left ventricular contractility, as measured by fractional area change, when compared with that in the other groups at both the 2 and 6 weeks time points (p < 0.05, CD56⁺CD34⁺CD144⁺ cells vs. CD56⁺CD34^–CD144^– cells and PBS). (D) No difference in left ventricular end-diastolic areas was observed between hearts injected with unsorted myoblasts and hearts injected with myogenic CD56⁺ sorted cells 2 weeks after cell transplantation. (E) The unsorted group also displayed a similar level of left ventricular contractility, as measured by fractional area change, as the CD56⁺ cell group.

(A) Representative images taken from transverse sections of the left ventricle stained by Masson's trichrome (muscle is stained red, collagen is stained blue). The hearts injected with CD56⁺CD34⁺CD144⁺ cells displayed smaller infarct scar areas than did the control hearts injected with phosphate-buffered saline (PBS) only. Scale bars = 1 mm. (B) Compared with all groups, the CD56⁺CD34⁺CD144⁺ cell-injected hearts had the smallest scar tissue area 2 and 6 weeks after infarction (*p < 0.05, CD56⁺CD34⁺CD144⁺ cells vs. PBS).

(A) Masson's trichrome staining (muscle is stained red, collagen is stained blue) at the peri-infarcted area of the CD56⁺CD34⁺CD144⁺ cell-injected heart 2 weeks after cell transplantation. Scale bar equals 500 µm. The dotted line shows the engraftment area of human muscle-derived cells, (B) which corresponds to fast skeletal myosin heavy chain-positive (green) immunostaining. In the image, the fast skeletal myosin heavy chain-positive myofibers are stained green, nuclei are stained blue, and cardiac troponin I-positive cardiomyocytes are stained red. Only the engraftment regions expressed fast skeletal myosin heavy chain; normal myocardium was negative for fast skeletal myosin heavy chain. Scale bar = 50 µm. (C) CD56⁺CD34⁺CD144⁺ myoendothelial cells regenerated more fast skeletal myosin heavy chain-expressing myocytes than did CD56⁺CD34^–CD144^– myogenic and CD56^–CD34⁺CD144⁺ endothelial cells (*p < 0.05). (D) A few nuclei (blue) found in the fast skeletal myosin heavy chain-positive (green) engraftment area stained positive for human-specific proliferating cell nuclear antigen (red, arrows). Scale bar = 33.3 µm. (E to L) Expression of a cardiac cell markers by myoendothelial cells in vivo. (E) Engrafted myoendothelial cells expressing the nLacZ reporter gene (blue, arrowheads) colocalized with (F) cardiac troponin I (red, arrowheads) staining. 4',6-diamidino-2-phenylindole-only (G, blue, arrow) and colocalized with troponin I (H, pink and blue). (I) An nLacZ-expressing donor cell (blue, arrowhead) colocalized with the (J) cardiac troponin T marker (green, arrowhead). DAPI-only (K, blue, arrow) and colocalized with troponin T (L, green and blue). Scale bars = 50 µm.

(A) Representative images of CD31 immunostaining in the peri-infarct and the infarct regions of hearts transplanted with CD56⁺CD34^–CD144^– cells, CD56^–CD34⁺CD144⁺ cells, and CD56⁺CD34⁺CD144⁺ cells. Scale bars = 100 µm. (B) Hearts transplanted with CD56⁺CD34⁺CD144⁺ cells display a higher capillary density within the infarct when compared with the other groups (*p < 0.05, CD56⁺CD34⁺CD144⁺ vs. CD56⁺CD34^–CD144^– and CD56^–CD34⁺CD144⁺; p < 0.05, all cell groups vs. phosphate-buffered saline [PBS]).

(A and B) Proliferating cardiomyocytes within the peri-infarct region were identified by colocalization of both Ki-67 (purple nuclear stain, arrow) and cardiac troponin I (cTnI) (brown cytoplasmic stain) within the peri-infarct region (arrow). Some of the proliferating cells stained by Ki-67 exist within the infarct zone, but do not colocalize with cTnI (arrowhead). Scale bars = 50 µm. (C) The number of endogenous proliferating cardiomyocytes was higher in the peri-infarct region of the heart injected with CD56⁺CD34⁺CD144⁺ cells compared with that seen in other groups at both 5 days and 6 weeks after cell transplantation (*p < 0.05, CD56⁺CD34⁺CD144⁺ cells vs. CD56⁺CD34^–CD144^– cells, CD56^–CD34⁺CD144⁺ cells, and phosphate-buffered saline [PBS]). (D) The number of apoptotic cardiomyocytes was measured at both 5 days and 6 weeks after myocardial infarction (p < 0.05, CD56⁺CD34⁺CD144⁺ cells vs. PBS). HPF = high-power fields; TUNEL = terminal dUPT nick end-labeling.

(A) An increase in vascular endothelial growth factor (Vegf₁₆₅) mRNA production by CD56⁺CD34⁺CD144⁺ cells was observed when cultured under hypoxia for 24 h in comparison with that seen in control normoxic conditions. In contrast, hepatocyte growth factor (Hgf), basic fibroblast growth factor (b-Fgf), and insulin-like growth factor-I (Igf-I) mRNA expression by human CD56⁺CD34⁺CD144⁺ cells either significantly decreased or was not detectable after 24 h of hypoxia. (B) All cell types greatly increased their secretion of Vegf mRNA after exposure to hypoxic culture conditions for 24 h when compared with control cells in normoxia culture conditions by quantitative polymerase chain reaction analysis of Vegf mRNA expression (*p < 0.01, normoxia vs. hypoxia). (C) These results were further confirmed by VEGF ELISA analysis (*p < 0.05, normoxia vs. hypoxia).