302 Chapter 12 and processed with a fixation kit (Parse Biosciences). Fixed samples were combined, barcoded, and sequenced according to the Parse barcoding protocol. Sequencing data was analyzed with the split-pipe software from Parse to obtain cell/gene expression matrices for each sample. Single-cell RNA sequencing analysis. Single-cell RNAseq (scRNAseq) data was analyzed using the Seurat package (version 4.0.5) in R (version 4.1.0). The scRNAseq data of all hCSs samples was combined and analyzed, and cells with at least 750 features were selected from three control and three PLN-R14del mutated samples. A reference data set from Grancharova et al.25 comprising hiPSC-CM cultures that were matured for 12, 24, or 90 days was used for mapping combined with the MapQuery function in Seurat (Supplementary Figure 4A-B). ScTransformed expression data was used to obtain variably expressed genes. RNA count data was regularly normalized and scaled with regression of mitochondrial gene expression levels and the number of expressed genes (nFeature). The normalized data was used to generate a PCA plot from the variable genes. PCA values were used to integrate cells of different samples via the harmony method (function: runHarmony25,2626). Harmony scores were used for dimensionality reduction of the cells using UMAP, and to cluster cells. Cell type categories were assigned to each cell cluster after examination of expressed marker genes. Differential gene expression was modelled and assessed using the glmQLFit functions in edgeR27 with added per-cell gene detection rate information as recommended by Soneson et al.28 Hereby, expression was modelled using a linear model that included cell type and PLN genotype information next to the gene detection rate. AAV Treatment. I-1c cDNA, under the control of the CMV promoter, was packaged in an AAV2i8.29 AAV.I-1c was prepared to a final titer of 4.73 × 10^14 viral genomes (VG)/mL. To determine the optimal multiplicity of infection (MOI), transduction with an MOI of 1000, 10.000, 100.000, and 500.000 was used in one-week-old PLN-R14del hCSs, whereafter the spheroids were cultured for two more weeks before harvested. The endogenous I-1C expression was determined by RT-qPCR while the Ca2+ handling function was determined by optical Ca2+ transient analysis of peak value, decay time, and beating rate parameters of the hCSs (Supplementary Figure 6). For the final experiments, hCSs were incubated with a MOI of 10.0000 in 100 μL media for 24 h. After 24 hours, 200 μL of the spheroid medium was added to each well. Every 2-3 days, half of the medium was replaced with maturation medium for 14 days post AAV transduction. Optical Ca2+ transient analysis. Ca2+ transient analysis was performed to evaluate Ca2+ handling properties in both control and PLN-R14del hiPSC-CMs with and without AAV treatments. Briefly, cells were loaded for 30 min in FluoroBrite DMEM Media (Thermo Fisher),
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