Abstract
Background: The ability to create human cardiomyocytes from induced pluripotent stem cells (iPSCs) has spurred advances in disease modeling and drug testing, but there remains an ongoing need to improve the purity and functional maturity of derived cardiomyocytes. Membrane proteins are an integral component of cell signaling and specification. To better understand iPSC-cardiomyocyte differentiation and identify potential surface markers of cell maturity, we set out to examine how membrane-targeted proteins gradually remodel in differentiating cardiomyocytes. Methods: Human iPSCs from three donors were differentiated into cardiomyocytes in 40 independent differentiation plates. We sampled protein expression at independent time points at up to daily intervals during day 0 to day 14 post-differentiation, then sparsely up to 100 days. The extracted proteins were analyzed using a Q-Exactive HF Orbitrap high-resolution mass spectrometer to quantify how the relative quantity of membrane-targeted proteins changes over time in differentiating cells. Results: We identified 1095 annotated cell membrane proteins and quantified their temporal changes in abundance across 40 samples. Protein expression in the first two principal components recapitulated gradual differentiation of iPSCs into mesoderm, cardiac progenitors, and more mature cardiomyocytes. Membrane protein expression partitioned the samples into three discernible clusters, representing early (day 0-5), mid (day 6-10), and late (day 11+) differentiation time points. In total, 115 cell membrane proteins showed differential expression in late vs. early differentiation, including two popeye-domain-containing proteins (POPDC2 and BVES) known to be involved in cardiac development. In particular, we identified two proteins, dysferlin (DYSF) and alpha-1 syntrophin (SNTA1), which continued to show strong upward changes in late differentiation time points, nominating these proteins for investigation as markers for mature cells. Conclusion: We report the time-resolved expression profiles of over 1000 membrane-targeted proteins during iPSC-cardiomyocyte differentiation. Future work into the membrane proteome may lead to maturity markers to guide maturation efforts.
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