Physical developmental cues for the maturation of human pluripotent stem cell-derived cardiomyocytes

Abstract

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications.

Figure 1

Schematic diagram illustrating developmental factors that potentially impact the in vitro maturation process from human pluripotent stem cell (hPSC)-derived cardiomyocytes to an adult-like phenotype with highly organized sarcomeres and intercellular junctions. This review focuses on physical developmental cues from electrical stimulation and mechanical loading, and also mentions factors including genetics, supporting cells and substrate, metabolism, and both circulating and membrane bound signaling molecules.

Figure 2

Representative images of hPSC and hPSC-CM. (A) Representative images of human pluripotent stem cells (hPSCs) (left), a monolayer culture of hPSC-derived cardiomyocytes (hPSC-CMs; unstained, middle), and dissociated and re-plated human embryonic stem cell-derived cardiomyocytes immunostained with antibodies against cardiac troponin T (TNNT2; right) [139]. (B) Cardiac troponin I (TNNI3) immunostaining of a monolayer culture of human induced pluripotent stem cell-derived cardiomyocytes at day 29 of differentiation showing random patterns of striations. (C) Immunostaining of a three-dimensional tissue strip with well-aligned troponin-stained hPSC-CMs. Green, TNNT2 labeling (A, C), TNNI3 labeling (B); blue, DAPI labeling.

Figure 3

Optical characterization of hPSC-CM electrophysiology. Optical mapping of enzymatically digested and re-plated human induced pluripotent stem cell-derived cardiomyocyte monolayers recorded 9 days (A-C) and 13 days (D-F) after re-plating. (A) Transmembrane voltage map of 9-day re-plated monolayer. (B) Activation time map and local conduction velocity vectors of (A). (C) Action potential recorded from location indicated by white box in (A). Electrical coupling among the cells of this 9-day old monolayer is poor, as indicated by the disorganized activation time map, rough wavefront of the propagating AP and slow conduction velocity (5.4 cm/second). (D) Transmembrane voltage map of a 13-day re-plated monolayer. (E) Activation time map and local conduction velocity vectors of (D). (F) Action potential recorded from location indicated by white box in (D). Electrical coupling was much improved with increased time in culture, as indicated by the near planar propagating action potential as well as faster conduction velocity (10.5 cm/second). Dashed lines in (C) and (F) indicate the time points of the corresponding transmembrane voltage maps in (A) and (D).