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Background: Transverse tubules (TT) are tunnel-like extensions of sarcolemma studded with ion channels coupling excitation, through the cytoplasm, to contraction in the sarcomere of matured cardiomyocytes (CMs). Expression timing of sub-cellular TT-related genes (TT-rgs) in individual human iPSC-derived CMs (hiPSC-CMs) has not yet been reported. Objective: Map out the gene program of TT-rgs by sub-cellular locations during hiPSC-CM differentiation using single-cell transcriptomics (scRNA Seq.). Methods: hiPSC-CMs were differentiated from 2 commercially available lines using sequential GSK3 and Wnt signaling inhibition. The %CMs were assayed by flow cytometry for sarcomeric myosin heavy chain protein, a CM biomarker. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. We categorized 73 TT-rgs by cellular location of the coded protein (i.e. sarcomere, cytoplasm or sarcolemma). The expression pattern for each location was categorized as induced, repressed, or neither based on the median transcript per million for each individual cell normalized to iPSCs. Results: CMs increased from Conclusion: Single-cell transcriptomes in differentiating hiPSC-CMs revealed a discordance between sarcolemma (mostly not expressed) and sarcomeric genes being induced. Defining timing and within cell variability of TT sub-cellular genes will be critical to understand human CM maturation.

Background: Adrenergic receptors (AR) in an individual cardiomyocyte (CM) are not uniformly expressed at the single-cell level (Myagmar et al., 2017). The timing and cellular distribution of AR signaling (ARS) genes in individual human iPSC-derived CMs (hiPSC-CMs) have not yet been reported. Objective: To map out the transcription program of ARS genes during hiPSC-CM myogenesis using single-cell transcriptomics (scRNA Seq.). Methods: 132 ARS in CM genes curated by the Kyoto Encyclopedia of Genes and Genomes were studied. The CMs were derived from 2 commercially available hiPSC lines using sequential GSK3 and Wnt signaling inhibition. The CMs were assayed by flow cytometry with sarcomeric myosin heavy chain (MYHC6/7) protein, a biomarker of myogenesis. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. Data from 10 somatic cell preparations and iPSCs defined the signal specificity and biological noise in the system. The expression patterns were categorized as induced, repressed, or neither based on the median transcript per million for individual cells normalized to iPSCs. A p -value of Results: The CMs had spontaneous contractions by D14 and increased from 90% of differentiating cells did. MYH6/7 median expression increased 15-fold from D14 to D30 (p Conclusion: Single-cell transcriptomes in derived hiPSC-CMs revealed a lack of uniformity between upstream ARS genes and early sarcomeric genes. Defining the timing and cell-to-cell variability of the ARS gene program will be critical to understanding its mechanistic relationship to the sarcomeric gene program in humans.