Figure 2.

Adoptive transfer of CD4⁺ Treg potentiates neonatal heart regeneration after cryoinfarction. (A) Schematic diagram showing the experimental design. (B) Flow cytometric analysis showing engraftment and infiltration of CD3⁺CD4⁺hCD2⁺ Treg in the spleen and myocardium, respectively, at day 7 following CI to a P3 heart of NOD/SCID mice. Quantification of (C) %Treg among total splenocytes or (D) absolute number of hCD2⁺ Treg per mg heart tissue at the indicated time points after adoptive transfer by flow cytometry, n=3 per time point. (E) Images of scar tissues, scale bars: 2000 um; and Masson's trichrome staining showing representative serial cross sections of fibrotic tissues in blue at 4 weeks post CI, scale bars: 1000 um. (F) Quantification of fibrotic tissue coverage based on (E), n=7. (G) Immunostaining on frozen sections for Ki67⁺ (red) or pH3 (red) and cTnT⁺ (green) cells within the border zone at day 7 post CI, scale bars: 50 um. Arrows indicate cardiomyocytes positive for Ki67 or pH3 and square denotes magnified images on the right. Quantification of absolute number of (H) Ki67⁺cTnT⁺ or (I) pH3⁺cTnT⁺ cardiomyocytes per mm² area, n=7. (J) Echocardiographic analysis and quantification showing (K) %fractional shortening or (L) %ejection fraction at 8 weeks post CI, n=7. (C, D, F, H, I, K, L) Data are presented as mean±S.E.M., *P<0.05, **P<0.01.