Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes

Abstract

The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells and human-induced pluripotent stem cells, has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models; however, finding ways to mature hPSC cardiomyocytes is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC cardiomyocytes, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC cardiomyocytes include long-term culturing, 3-dimensional tissue engineering, mechanical loading, electric stimulation, modulation of substrate stiffness, and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature's diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.

Keywords: disease modeling; human pluripotent stem cell–derived cardiomyocytes; maturation; pharmacologic screening.

Fig. 1

Morphological differences between an immature hPSC-CM and adult rat cardiomyocyte. A (hPSC-CM) and B (adult rat): Overview of contractile cytoskeleton with alpha-actinin staining (green) and blue nuclear counterstain. C (hPSC-CM) and D (adult rat): cellular ultrastructure by electron microscopy. Note that there are significant differences with respect to cell size, length to width ratio, mitochondria quantity, size and morphology, appearance of T-tubules (arrows), and elongated nuclei. (Scale bar in A and B: 25 μm, C and D: 0.2 μm) Fig. 1B and 1C were kindly provided by Scott Lundy and Dr. Michael A. Laflamme.

Fig. 2

Effects of long-term culture on hPSC-CM maturation. In Panel 1A and 1B, representative hPSC-CMs immunostained for alpha-actinin (red) and filamentous actin (phalloidin, green). Nuclei (blue). C–F. The multinucleation cell percentage, sarcomere length, and cell area were significantly increased with a decrease in cell circularity. G. Electrophysiologically, older cardiomyocytes show significantly enhanced action potential upstroke velocity, a hyperpolarized maximum diastolic potential, and an increase in transient outward rectifier potassium currents. Panels A through F were modified from Lundy et al with permission from Dr. Michael A. Laflamme. Panel G was modified from Sartiani et al with permission from Dr. Marisa E. Jaconi.

Fig. 3

Tissue engineered hPSC-CMs collagen constructs were subjected to a series of stretches to increase resting tension. Note the increased twitch heights at higher preload (insets). (B) Starling curves showing a linear increase in active force in response to increased diastolic length. This figure was reproduced from Tulloch et al with permission.

Fig. 4

Potential application of hPSC-CMs. hPSC-CMs can be used and already show promising results in regenerative medicine. These cells are also valuable for disease modeling and pharmacological studies, as discussed in the main text. Enhancing hPSC-CM maturation state may allow for improved therapeutic applications. As we develop methods to generate cardiomyocytes of different maturation states, we will be able to gain significant insights into human cardiac development.

Fig. 5

Summary of current maturation approaches and phenotypes achieved so far. A and B: representative immature hPSC-CMs and intermediate hPSC-CMs, respectively. The cells were stained for alpha-actinin (green) and filamentous actin (phalloidin, red). Nuclei (blue). Elongated cell shape, more organized sarcomere structures, longer sarcomeres, and more abundant mitochondria were achieved with manipulations. Compared with immature hPSC-CMs, the intermediate cardiomyocytes display higher conduction velocity, contractile force, and increased calcium transient kinetics. Scale bars in A and B: 25 μm, C and E: 0.5 μm, D: 5 μm) Panel B, C, D and E were modified from Lundy et al with permission from Dr. Michael A. Laflamme.