309 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 Figure 3. Contribution of Cardiac Fibroblast (CF) numbers to hCSs size. (A) Representative cardiac spheroid morphology at D2, D7, D14, and D21 after the controlled generation of hCSs containing hiPSC-derived fibroblasts (hiPSC-CFs) and hiPSC-CMS, both derived from the PLN-R14del (D4iR14del) line in the ratios 100% hiPSC-CFs (top) to 100% hiPSC-CMs (bottom) and 5 different ratios in between. Scale bar: 400 μm. (B) Bar graphs of the size quantification from C31iCTR (CTR) spheroids on day 2 and day 21 post-hCS generation. (C) Bar graphs of the size quantification from D4iR14del spheroids on day 2 and day 21 post-hCS generation. Biological replicates = 1, technical replicates 5-8 hCSs per group. * P< 0.05 - **** P < 0.0001 vs. hiPSC-CF only group by one-way ANOVA. Single-cell sequencing reveals cellular diversity and PLN-R14del phenotype pathways To investigate the cardiac spheroid composition and functional heterogeneity, we performed split-pool ligation-based transcriptome sequencing (SPLiT-seq) of 760 single cells from PLN-R14del spheroids (total 386 cells) and healthy or isogenic corrected spheroids (total 374 cells) (Figure 4A, Supplementary Figure 5). Based on previous clustered mapping25 (Supplementary Figure 5A-B), we partitioned these cells into three clusters, which were annotated as cardiomyocytes group 1 (CM1), cardiomyocytes group 2 (CM2), and fibroblasts (fibro) (Figure 4B, Supplementary Figure 5C). We found that the healthy CTR spheroids were mainly composed of CM1 and CM2 grouped cells (Supplementary Figure 5D). The CM1 cluster is distinguished from the CM2 group by the abundance of genes; deleted in colorectal cancer (DCC), a large transmembrane receptor of netrin-1, critical for mediating netrin-1’s cardioprotective function and sodium leak channel non-selective channel protein (FAM155A)
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