Non-Cardiomyocytes Influence the Electrophysiological Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes During Differentiation

Abstract

Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca 2+ ) imaging, and intracellular recording techniques, we found that intracellular Ca 2+ -cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca 2+ -handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to infl uence early cardiac development. Our study is the fi rst to demonstrate that non-cardiomyocytes infl uence electrophysiological maturation of early hESC-CMs in cultures. Defi ning the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors

“…When the embryonic mouse heart develops from a "primitive heart tube" at E8.5 to a well-defined tubular shape expanded at the ventricular end at E9, Ca 2+ activity increases from 50% to almost 100% of the nascent myocytes (35) and electrophysiological maturation of human ES-derived cardiomyocytes involves the development of more rapid spontaneous Ca 2+ transients (36). In comparison, the ETS2/MESP1 reprogrammed cells appear to be at the early to intermediate stages of cardiomyocyte maturation.…”

Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue

“…Color images available online at www.liebertpub .com/tea These findings are consistent with previous studies showing that fibroblasts reduce the intrinsic beating frequency of cardiomyocytes, and nonmyocytes play a role in modulating the electrophysiological maturation of stem cellderived cardiomyocytes during differentiation. 44,45 The type of stromal cells in cocultured constructs influences their interactions with cardiomyocytes and matrix remodeling Different populations of ''stromal cells'' or ''fibroblasts'' have drastic differences in their phenotype, gene expression, and signaling. 46 To understand how stromal cells from different sources affect remodeling and cellular alignment in cardiac constructs, we generated cardiac constructs by coculturing cardiomyocytes with four different fibroblast or stromal cell lines in low-density collagen for 4 days.…”

Cardiac muscle regeneration: lessons from development

“…Given the utility of applying developmental insights, additional research is needed to fill the gaps in myocardial development that are most pertinent to stimulating proliferation and enhancing differentiation of adult cardiac stem and progenitor cells. Much is known about creating cardiogenic progenitors during development, but less is known about the signals that subsequently convert committed cells into cardiomyocytes themselves, and nearly nothing is known about mechanisms that drive myocyte maturation, including acquisition of the adult electrical and physical properties characteristic of the heart as a biomechanical pump, although interactions with nonmyocytes are important clues to the latter (Narmoneva et al 2004;Kim et al 2010). Accelerated progress will undoubtedly come from applying high-throughput technologies to the comprehensive screening of signaling molecules and pathways for cardiogenic activity using hESCand hiPSC-derived progenitors, and even using isolated progenitors from adult heart, followed by application of these novel signals and pathways to restoring heart function.…”