244 Chapter 9 aggregated and pre-processed using SAVER41 to impute missing data prior to downstream analysis. The Seurat package 2.0 implemented in R was used to perform normalization, unbiased clustering, t-distributed stochastic neighbor embedding (t-SNE) visualization, and differential gene expression analysis as described in the tutorials (http://satijalab.org/ seurat/)42. Briefly, the aggregated cell count matrix was first normalized by dividing the number of UMI for each transcript by the total UMI for the cell, multiplying by 10,000, and log-transformed. Highly variable genes were selected using the FindVariableGenes function in Seurat and used as input for principal component analysis (PCA). Based on the first 20 principal components, cell clusters were identified based on their PCA score. To visualize cells in a high-dimensional space, two-dimensional projections created by t-SNE. To assign identities to these subpopulations, we cross-referenced their marker genes with known cardiac subtype markers from the literature. To detect differentially expressed genes between wild-type and PLN R14del iPSC-CM sub-populations, we performed pairwise comparisons using the nonparametric Wilcoxon rank-sum test through the FindMarkers function. An adjusted P-value (Bonferroni correction) cut-off < 2×10−6 was used to identify differentially expressed genes. Three-dimensional Engineered Heart Tissues (3D-EHTs). The 3D-EHTs were generated in agarose casting molds using solid silicone racks (EHT Technologies) as described with modifications43. Briefly, about 1x106 iPSC-CMs were suspended in a fibrin hydrogel (100 μL total) composed of 10 μL Matrigel (Corning), 5 mg/mL bovine fibrinogen (2.53 μL of 200 mg/ mL fibrinogen reconstituted in 0.9% NaCl) and supplemented with 0.1 mg/mL aprotinin (Sigma Aldrich) and 3 U/mL thrombin (Sigma Aldrich). Once polymerized, the silicone racks with the newly formed fibrin gels were transferred to a new 24-well plate and cultured for 3-4 weeks in culture medium consisting of DMEM:RPMI media (1:1) supplemented with 0.25% dialyzed fetal bovine serum (JR Scientific), 0.5x B27 supplement (Gibco), 5% knock-out serum replacement (Gibco), 1% penicillin/streptomycin (Gibco), and 33 μg/mL aprotinin. The culture medium was refreshed every 3-4 days. Videos of the deflecting posts were recorded at 75 frames/s using the SI8000 Cell Motion Imaging System (Sony), at baseline and 72 hours following BiX treatment. The video recordings were processed by MuscleMotion44 to quantify the contraction amplitudes. Absolute force values were derived from calibrated measurements of post displacement considering an elastic modulus of 1.7 MPa, a post radius of 0.5 mm and a distance between posts (length) of 10 mm.37 High-throughput contractility analysis. The hiPSC-CMs were plated on Matrigel-coated surfaces at a density of 20,000 cells per well in a 384-well plate (Greiner Bio-One) in 100 µL of replating media (RPMI-B27, 10% knock out serum replacement). The replating media was gradually transitioned to RPMI/B27 by replacing 50% of the media every 2 days for 6-8 days prior to analysis. For siRNA-mediated knockdown, hiPSC-CMs were transfected with
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