Fig. 4

Cellular and molecular assessment of 3D cardiac microtissue. a Immunofluorescent staining for cardiac-specific marker (sarcomeric-alpha actinin; SAA) and MSC/pan-fibroblast-specific marker (vimentin; VIM) to visualize cell distribution in CM-MSC cardiac microtissue on day 14. Nuclei were stained with DAPI (blue). Scale bars, 50 μm. b Cardiac microtissue sections were co-immunostained with anti-vimentin (green) for MSC/pan-fibroblast-specific marker and anti-DDR2 (red) for the cardiac fibroblast marker (left panel). Cells positive for both vimentin/DDR2 occurred within cardiac microtissue (arrows), suggesting a transdifferentiation of MSCs into fibroblasts. hESC-derived MSCs were analyzed by immunofluorescence using anti-vimentin (green) and anti-DDR2 (red) (right panel). c Principal component analysis (PCA) based on total gene expression in undifferentiated hESC (n = 3), hESC derived CMs (hESC-CM) (n = 2), CM-MSC cardiac microtissue (hESC-CM + hESC-MSC) (n = 3), and human heart (n = 1). d Reactome pathway terms enriched in up-regulated DEGs (> 2-fold change) in the transcriptome of CM-MSC cardiac microtissues, compared to that of hESC-CM. e qRT-PCR analysis of cardiac function-related genes, including SGCD, MYL1, SCN7A, SCN1B, KCNJ2, and KCNE4. Data are the means±SD of three independent experimental replicates (n = 3).**p < 0.01