Fig. 5

Recapitulation of phenotypes observed in TGF-β1-induced cardiac fibrosis using 3D cardiac microtissue. a Representative images of CM-MSC cardiac microtissue with or without 5 ng/ml TGF-β1 treatment for 2 weeks (left panel). Representative trace obtained by plotting z-axis profile of cardiac microtissue beating (right panel). b Masson’s Trichrome staining of CM-MSC cardiac microtissue sections at serial time points after TGF-β1 treatment. Note the extensive interstitial fibrosis represented by the blue stains. Scale bars, 100 μm. c Immunofluorescent staining of apoptotic CMs in cardiac microtissue with an apoptosis-specific marker (Cleaved caspase 3; Cl-Casp3) and cardiac specific marker (sarcomeric-alpha actinin; SAA). White arrow indicates cells co-stained with SAA and Cl-Casp3. d Percentage of apoptotic CMs by quantifying ratio of Cl-Casp3 positive cells per number of DAPI-stained cells. Data are the means±SD of replicates (n = 3). **p < 0.01. e Average diameters of cardiac spheroids. Data are the means±SD of replicates (n ≥ 11). **p < 0.01. f Immunoperoxidase staining of myofibroblast-specific marker (alpha-smooth muscle actin; α-SMA) in cardiac microtissues after TGF-β1 treatment. g CM-MSC cardiac microtissue sections were co-immunostained with anti-vimentin (green) and anti-α-SMA (red). Cells undergoing fibroblast-to-myofibroblast transdifferentiation were positive for both vimentin and α-SMA (arrows). Nuclei were stained with DAPI (blue). Scale bars, 50 μm