Learn from Your Elders: Developmental Biology Lessons to Guide Maturation of Stem Cell-Derived Cardiomyocytes

Abstract

Human pluripotent stem cells (hPSCs) offer a multifaceted platform to study cardiac developmental biology, understand disease mechanisms, and develop novel therapies. Remarkable progress over the last two decades has led to methods to obtain highly pure hPSC-derived cardiomyocytes (hPSC-CMs) with reasonable ease and scalability. Nevertheless, a major bottleneck for the translational application of hPSC-CMs is their immature phenotype, resembling that of early fetal cardiomyocytes. Overall, bona fide maturation of hPSC-CMs represents one of the most significant goals facing the field today. Developmental biology studies have been pivotal in understanding the mechanisms to differentiate hPSC-CMs. Similarly, evaluation of developmental cues such as electrical and mechanical activities or neurohormonal and metabolic stimulations revealed the importance of these pathways in cardiomyocyte physiological maturation. Those signals cooperate and dictate the size and the performance of the developing heart. Likewise, this orchestra of stimuli is important in promoting hPSC-CM maturation, as demonstrated by current in vitro maturation approaches. Different shades of adult-like phenotype are achieved by prolonging the time in culture, electromechanical stimulation, patterned substrates, microRNA manipulation, neurohormonal or metabolic stimulation, and generation of human-engineered heart tissue (hEHT). However, mirroring this extremely dynamic environment is challenging, and reproducibility and scalability of these approaches represent the major obstacles for an efficient production of mature hPSC-CMs. For this reason, understanding the pattern behind the mechanisms elicited during the late gestational and early postnatal stages not only will provide new insights into postnatal development but also potentially offer new scalable and efficient approaches to mature hPSC-CMs.

Keywords: Cardiomyocyte maturation; Human embryonic stem cells; Human induced pluripotent stem cells; Postnatal cardiac development.

Figure 1.. Human embryonic heart development.

The primitive heart tube forms by human embryonic day 22 (E22) and is divided in four different regions: from head to tail, the truncus arteriosus will give rise to the aorta and the pulmonary trunk; the bulbus cordis will develop into the right ventricle; the ventricle region will form the left ventricle and the anterior portion of the right and the left atria; and the sinus venosus develops into the posterior portion of the right atrium, the SA node, and the coronary sinus. By E24, the heart tube starts to bend in a primitive C-shape. Ventricular myocytes undergo trabeculation, whereas the endocardial cells start to migrate into the endocardial cushions. By E28, the heart tube creates a S-shape with the primordial division of the four chambers. By E56, the foetal heart is completely formed; the septa primum and secundum divide the two atria, which remain connected through the foramen ovale, while the endocardial cells from the cushions give rise to the tricuspid and the mitral valves. The dashed line indicates the coronal sections depicted below each stage.

Figure 2.. Cardiomyocyte maturation.

Fetal cardiomyocytes (CMs) are small and round-shaped, with undeveloped mitochondria and sarcomeres. Multiple environmental cues, such as mechanical and electrical stimuli, extracellular matrix interactions, and interactions with non-cardiomyocytes drive gradual maturation of CMs. Through neonatal and adult stages, CMs become elongated and display increased cytoskeletal organization. Expression of Connexin-43 and N-cadherin increases, and mitochondria develop mature cristae. Adult CMs are also often binucleated and display robust T-tubules and intercalated disks.