Title Physical developmental cues for the maturation of humanpluripotent stem cell-derived cardiomyocytes

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. Introduction Human pluripotent stem cells (hPSCs) of embryonic (embryonic stem cells (ESCs)) or experimental (induced pluripotent stem cells (iPSCs)) origin [1-5] represent the most viable cell source for in vitro generation of large numbers of cardiomyocytes (CMs). The directed differentiation of hPSCs to CMs has led to important research advances, including innovative platforms for the study of human development and for disease modeling. It has also reaffirmed the promise of cardiac regenerative medicine with immunologically compatible cells. To date, research has focused justifiably on cellular and molecular mechanisms that control induction, differentiation, proliferation and scalability of CM production [6,7]. These efforts have led to CM differentiation protocols ranging from monolayer to cell aggregate systems with diverse chemical additives (for example, bone morphogenic protein and * Correspondence: bohelerk@hku.hk University of Hong Kong, Stem Cell and Regenerative Medicine Consortium, Hong Kong, SAR, China Division of Cardiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Full list of author information is available at the end of the article © Zhu et al.; licensee BioMed Central Ltd months following its publication. After this time License (http://creativecommons.org/licenses/b medium, provided the original work is properly (http://creativecommons.org/publicdomain/zero 2014 activin agonists versus Wnt inhibitors) and a variety of culture techniques (plate, flask, bioreactor) [6,7] that can be employed for basic cell biology analyses [8,9], generation of engineered tissue constructs [10-13], and testing of regenerative potential after transplantation in experimental models of heart failure [14]. Despite these advances, a major hurdle for the experimental and clinical use of these cells has been their phenotypic ‘immaturity’ in vitro. In contrast to native adult CMs, hPSC-derived CMs (hPSC-CMs) are characterized as having small size and low capacitance, absence of T-tubules, lack of well-formed sarcomeres, poor overall calcium storage and handling, absence of multinucleation, relatively low numbers of mitochondria, metabolic dependence on glycolysis, and innate automaticity [15-18]. When transcriptionally compared to native human cardiac muscle cells, hPSC-CMs most closely resemble cells from embryonic or fetal heart [19]. Developmentally, physical cues and environmental factors are paramount for the production of structurally and physiologically mature CMs. In vivo, mechanical cues from the cell surface (cell-cell and cell-extracellular matrix . The licensee has exclusive rights to distribute this article, in any medium, for 12 , the article is available under the terms of the Creative Commons Attribution y/4.0), which permits unrestricted use, distribution, and reproduction in any credited. The Creative Commons Public Domain Dedication waiver /1.0/) applies to the data made available in this article, unless otherwise stated. Zhu et al. Stem Cell Research & Therapy Page 2 of 18 2014, 5:117 http://stemcellres.com/content/5/5/117 (ECM) interactions) are converted into intracellular signals that can lead to phenotypic changes through a process known as mechanotransduction (reviewed in [20-24]). In fact, mechanical stretch and electrical activity are extremely potent biological cues that, in the heart, not only regulate contraction amplitude on a beat-by-beat basis, but also drive mechanical and electrical remodeling of the myocardium during cardiac development, hypertrophy, and disease. Heart tissues and individual myocardial cells experience not only self-generated mechanical force, but also passive and active stretching, all of which can activate mechanotransduction pathways. These physical cues require contact-dependent interactions of cells with ECM, with other cells, as well as with physical features associated with their environment (that is, topography). The fibrous topography is associated with anisotropic alignment of CMs within the heart [25] and gives rise to distinct longitudinal and transverse structures, allowing for directionally distinct pathways of force recognition and transmittance [26,27]. Indeed, static transverse and longitudinal loading can differentially activate stress-induced mitogen-activated protein kinase (MAPK) [28] and alter cardiac-specific protein secretion [29], while anisotropic mechanosensing with focal adhesion kinase (FAK) phosphorylation has also been reported in neonatal rat ventricular myocytes (NRVMs) [30]. Despite these scientific advances, much less is known about the events that take place in developmentally ‘immature hPSC-CMs’. Here, we review structural components and physical stimuli that may influence hPSC-CM developmental maturation in vitro, and discuss data from animals and humans to describe known mechanisms. We focus on mechanical and electrical factors, and touch upon metabolic and hypertrophic signals that have been implicated in the adaptation of hPSC-CMs in two dimensions and, where available, cells engineered in three dimensions. We illustrate how in vitro differentiated hPSC-CMs can respond to some of the same physical cues present in embryonic, fetal and adult heart but point out that these factors are preferably interpreted in a three-dimensional context that can be recapitulated in vitro. We also highlight areas that are currently not well represented in published works, including the interactions with nonmyocytes and application of transcriptomics to electrical and mechanical transduction events in hPSC-CMs. By shedding light on these areas, future research advances may overcome current limitations of hPSC-CMs for reliable disease modeling, drug discovery, cardiotoxicity testing and studies of developmental maturation. Heart development and physical cues Heart development and cell growth involve dynamic interactions between genetic and epigenetic or environmental factors [31] in a spatially complex manner [32]. In response to transcriptional cascades and morphogen gradients, cells in the lateral plate mesoderm and secondary heart fields commit to the CM lineage and presumably acquire an epigenetic phenotype that impedes or prevents de-differentiation. In the embryo and early fetal stages, fully committed CMs increase organ mass mainly through an increase in cell numbers (hyperplasia). During subsequent fetal and perinatal stages, mass increases through hyperplasia and through increased cell size and volume (hypertrophy). Post-natally, increases in cardiac mass occur almost exclusively by hypertrophy. During the perinatal period, hemodynamic loads increase dramatically as the heart assumes its primary role as a circulatory pump. Altered mechanical stimuli include a marked pressure increase and large pulsatile volume changes. In response to the increased work load and energy demand, mitochondrial numbers increase, mitochondrial oxidative metabolism is up-regulated as fatty acids become available, while glycolytic metabolism becomes downregulated [33]. The major changes in cell phenotype and function including maturation of the sarcoplasmic reticulum, reversal of the (initially negative) forcefrequency relationship [34], multinucleation, and the localization of gap junctions to intercalated discs at cell termini all occur during the perinatal and early childhood periods. Heart rate, blood pressure, and diastolic stiffness continue to change throughout adolescence and into adulthood and old age. In the adult, the myocardium consists of myofibrils composed of rod-shaped CMs located next to fibroblasts and blood vessels, and these cells are held in place by the ECM and physical interactions with other CMs. The development and maturation of CMs from the fetal to adult stages of life rely on a balance between extrinsic and intrinsic mechanical loads that regulate protein synthesis, sarcomere assembly, cell size, contractile activity, and interactions with other cells and the ECM [35], which together ultimately determine the geometry and pump function of the heart. Mechanical forces have a continuous and crucial regulatory role in cardiogenesis, cardiac growth, development and maintenance. In the developing mammalian heart, two types of contractile or intrinsic mechanical activity are observed that are believed to involve actin-myosin interactions. The first, which is beyond the scope of this review, is cytoskeletal contraction, a process that drives morphogenesis and cardiac looping [31]. The second is associated with the rhythmic contraction of heart muscle with each cardiac cycle, which subjects CMs to c