REVIEWS Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine Elaheh Karbassi1,2,3, Aidan Fenix1,2,3, Silvia Marchiano1,2,3, Naoto Muraoka1,2,3, Kenta Nakamura1,2,4, Xiulan Yang1,2,3 and Charles E. Murry 1,2,3,4,5* Abstract | Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review , we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues with electromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine. 1Institute for Stem Cell and Remarkable progress has been made over the past disease. For example, hPSC-derived liver cells might not Regenerative Medicine, decade in our ability to control the differentiation of produce albumin or might lack the enzymatic capacity University of Washington, human pluripotent stem cells (hPSCs). Lessons learned to metabolize urea or drugs. hPSC-derived β-cells might Seattle, WA, USA. from studies on embryonic development have enabled not secrete insulin in response to a glucose challenge, 2Center for Cardiovascular hPSC differentiation to be directed into the ectoderm, whereas hPSC-derived neurons might lack spontane- Biology, University of endoderm and mesoderm lineages, and our knowledge ous firing, and late-differentiating neural cells, such as Washington, Seattle, WA, USA. of the distal branches of these germ layers is growing. oligodendrocytes, are still difficult to obtain. With the use of hPSCs we have learned about human These limitations are relevant for heart research and 3Department of Pathology, University of Washington, development, how to build tissues and how genetic var- therapy development. Cardiac drug development has Seattle, WA, USA. iants cause disease. Hopes are high that soon we will be slowed over the past 20 years, creating a large unmet 4Division of Cardiology, able to discover new drugs with the use of hPSCs and, need. Many cardiac genetic diseases have middle-age Department of Medicine, perhaps one day, use these cells in cell-replacement onset and are difficult to model with hPSC-derived University of Washington, therapies. cardio myocytes (hPSC-CMs). For cell-replacement Seattle, WA, USA. Building on these achievements, the next challenge is therapies, the electrical immaturity of hPSC-CMs might 5 Department of to understand and control cell maturation. Most proto- underlie the ventricular arrhythmias that accompany Bioengineering, University embryonic stages fetal stages 1 of Washington, Seattle, cols generate cells at or early , cell engraftment in animal models . Moreover, unlike WA, USA. typically stages just after organogenesis completion. studies of cell-lineage determination, we cannot rely on *e-mail: [email protected] Therefore, the generated cells lack many attributes of lessons from developmental biology to guide the matu- https://doi.org/10.1038/ adult cells that are desirable for drug screening, mod- ration of hPSC-CMs (BOx 1). Our knowledge of cardiac s41569-019-0331-x elling of adult-onset diseases or replacing cells lost to development at late gestation is limited2,3 and stems NATURE REVIEWS | CARDIOLOGY VOLUME 17 | JUNE 2020 | 341 REVIEWS Key points Properties of maturing cardiomyocytes Morphology. The growing heart undergoes substan- • Cardiomyocytes can be generated in vitro from stem cells with high throughput and tial morphological changes at the cardiomyocyte level. purity at a clinically relevant scale, although their differentiation status resembles an Growth of the prenatal heart is predominantly driven by embryonic state. cardiomyocyte proliferation (hyperplasia). In the first • Cardiomyocyte maturation entails adoption of multiple complex phenotypes, and a week of postnatal life in rodents, and during the first dec- number of methods to mature stem cell-derived cardiomyocytes have been successful ade in humans, cardiomyocytes undergo mitotic quies- in driving the cells towards a postnatal state. cence, and further growth of the heart occurs principally • Stem cell-derived cardiomyocyte phenotypes have been characterized with the use by increasing cardiomyocyte size14,15. The developing of global systems approaches, which has uncovered novel regulators and insights for maturation. cardiomyocyte coalesces with adjacent cardiomyocytes to assemble an electromechanical functional syncytium • Moving forward, strategies for cardiomyocyte maturation will require indication- specific optimization for intended applications of stem cell-derived cardiomyocytes, through well-characterized gap junctions and adhesive leveraging an optimal maturation state while utilizing combinatorial approaches. junctions that form the myocardium. Standard dif- ferentiation of hPSC-CMs in in vitro culture lacks the dynamic physical and environmental cues necessary to principally from studies in animal models. Although induce the degree of physiological hypertrophy observed a few pioneering studies on human late prenatal or in vivo from postnatal stages into adulthood, even after early postnatal heart growth have been performed4–6, prolonged culture16–18. As such, use of standard cell cul- much of what we know about human heart maturation ture protocols results in heterogeneous cell populations is formed on the basis of findings in vitro and in adult of small, misaligned, immature cardiomyocytes of varied hearts. Therefore, our mechanistic understanding of shape, generally lacking the well-formed myofibrils and cardiomyocyte maturation is not as advanced as that T-tubules, polyploidy, polarized intercalated discs or of embryonic development. abundant mitochondria seen in vivo19 (Fig. 1; TABle 1). Postnatal developmental changes in cardiomyocytes The adult cardiomyocyte in vivo has a large, aniso- are remarkable. The cells withdraw from the cell cycle, tropic, rod-like shape that is approximately 150 µm in typically undergoing one more round of DNA synthesis length, 20 µm in width, 15 µm in height and 40,000 µm3 without cell division, generating large, tetraploid nuclei in volume20. By contrast, cultured hPSC-CMs range (in humans) or binucleated myocytes (in rodents)6,7. The from circular cells with diameters of 5–10 µm and cardiomyocytes then grow massively, with 10-fold to heights of ~5 µm at the start of spontaneous beating, Pluripotent stem cells 20-fold increases in cell volume (hypertrophy)6. With the to oblong cells measuring 30 µm in length, 10 µm in Stem cells with the capacity to onset of lactation, fatty acid oxidation becomes the major width and 2,000 µm3 in volume after prolonged culture21. differentiate into any cell type source of ATP in cardiomyocytes, and mitochondria pro- Adult cardiomyocyte morphology not only provides the of the embryo. This term 8 encompasses embryonic stem liferate to occupy ~30% of the cell volume . Much of the structural framework of the cell but also directly estab- cells and induced pluripotent growth in cell size is due to the increase in the contractile lishes other critical functional properties of the cell such stem cells. machinery, which consists of highly aligned myofibrils as electrophysiology and contractility. For example, with sarcomeres in a precise arrangement9. The reper- membrane capacitance is directly proportional to cell sur- Embryonic stages In humans, the first trimester toire of ion channels in the sarcolemma is remodelled, face area, such that smaller cardiomyocytes have lower of in utero development. such that automaticity is lost everywhere but in special- impulse propagation velocity and a reduced maximal ized pacemaking centres (such as the sinoatrial and atrio- action potential upstroke velocity than larger cells22,23. Fetal stages ventricular nodes)10,11. Simple caveolae in immature cells The elongated, anisotropic shape results in a high In humans, the second and undergo deep invagination to the core of the maturing length to width ratio that enables the presence of long third trimesters of in utero development. cell, creating the T-tubules that bring excitation stimuli to myofibrils with laterally registered sarcomeres, grant- the contractile apparatus12. Intercellular junctions, orig- ing efficient