br Limitations Adenoviral infection was

Limitations Adenoviral infection was performed 7days before FACS analysis. Due to constraints inherent in the hiPSC-CM differentiation system, the efficiency of adenoviral infection was not 100%. The percentages of the specific sub-populations reported do not take into account false negatives and only represent the percentages of each population that were recovered during sorting. We did not isolate MLC-2a+ or MLC-2v+ single positive populations through the simultaneous use of two distinct reporter genes due to concerns about competition between two strong cellular promoters and the potential for a sizable population of singly infected iPSCs. Consequently, analysis of double positive cells relied on antibody staining and the relative sensitivities of the MLC-2a and MLC-2v ARCA is unknown. Early ventricular CMs (MLC-2v+) were compared to the total MLC-2a+ population which, although enriched for developing atrial CMs, is contaminated with MLC2a+/MLC-2v+ ventricular cells. The action potential and calcium transient data presented represent the population average for each sorted population and we cannot rule out differences in action potentials and calcium transient morphologies between the two subsets of MLC-2a+ cells (MLC-2v+ or MLC-2v−). We suspect that the MLC-2a promoter would become more specific and the number of double positive cells would decrease as cultures more fully differentiate through increased time in culture or improved culture conditions. However, this was not tested directly. Many atrial or ventricular-specific markers become restricted to a single chamber late during cardiac development. Due to the early developmental phenotype of hiPSC-CMs, comparison of atrial and ventricular specific markers between the sorted populations was limited to markers with well characterized, early differences in atrial and ventricular expression.
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