Genetic Selection of Cardiomyocytes and Pacemaker Cells Derived From Human Embryonic Stem Cells

Abstract

Cardiomyocytes derived from human pluripotent stem cells are poised to transform heart disease treatment. However, current methods to produce stem cell-derived cardiomyocytes result in a mixture of cardiac phenotypes that is neither well controlled nor completely understood. Before safe and effective cardiac cell therapies can transition from the lab to the clinic, the consequences of phenotype heterogeneity need to be further studied. Improved genetic tools to label, track, and isolate specific cell types from heterogeneous differentiating populations are needed to understand the timing and identity of the signals that control stem cell differentiation. Traditional genetic manipulation methods do not translate well to human pluripotent cells. While viral methods have been useful, they are limited by random copy number and insertion site(s), insertional mutagenesis, transgene or endogenous gene silencing, and limited transgene capacity. To overcome these drawbacks, we used zinc finger nuclease gene targeting technology to create a novel line of "stoplight" human embryonic stem cells. "Stoplight" cells can be used to permanently mark and purify a subpopulation of differentiating cells based on the activation of a user-specified genetic promoter. We isolated near-homogenous populations of fluorescently marked undifferentiated cells and used the putative pan-striated muscle MCK/CK7 promoter to isolate a nearly homogeneous population of stem cell-derived cardiomyocytes using the "stoplight" cell line. Next, we used the "stoplight" cells to isolate and characterize two subpopulations of stem cell-derived cardiomyocytes that activate either the chicken GATA6 or MLC2v promoter during differentiation. We measured the automaticity, beating rate, action potentials, net ion currents and immunophenotype of these two populations and demonstrate they are distinct from each other. Our data suggest the populations represent putative early stage AV nodal (cGATA6) or ventricular (MLC2v) cardiomyocytes. Characterization of the bulk automaticity and beating rates of purified cGATA6 and MLC2v cardiomyocyte aggregates revealed significant differences that highlight the influence of subtype purity on bulk electrical behavior. Aggregates of cGATA6 cells beat significantly faster and maintained automaticity better than aggregates of MLC2v or admixed cardiomyocytes. In the past, stem cell-derived cardiomyocyte subtypes could only be studied as single cells; our data suggest the "stoplight" cell line is an ideal platform to study stem cell-derived cardiomyocyte subtypes and highlight the importance of subtype purity in bulk automaticity. As a whole, these studies highlight the importance of selecting subpopulations of differentiating cells. Genetically modified cells, such as the "stoplight" cell line, are a crucial stepping-stone to finding the signaling molecules and/or cell surface markers that will translate these subpopulations to clinical use. Safe cardiac cell therapies demand a detailed understanding of the automaticity of transplanted cells. Subtype purity has a dramatic effect on bulk automaticity and will be a critically important consideration in future therapeutics.