Cardiovascular disease (CVD) remains the leading cause of death in the United States with a range of treatments that vary according to the function that is comprised and patient-case severity. Despite progress in medicine and biomedical research, current cellular therapies are incapable of repairing and restoring cardiac function for heart-related CVDs that stem from dysfunctional cardiomyocytes (CM) or cell death. Since the human heart is incapable of regenerating itself naturally, a possible therapeutic strategy is to use human induced pluripotent stem cells (hiPSCs) to derive autologous CMs for replacing nonfunctioning or diseased cells. However, producing sufficient quantities of functionally suitable contractile and pacemaking CM subtypes poses a fundamental hurdle. Cellular interactions with the extracellular matrix (ECM) have been shown to transduce critical signals for cell-lineage specification. Previous studies that investigated the interactions between hiPSC-derived CMs and ECM proteins have shown that protein composition provides biochemical cues that are responsible for phenotypic maintenance and development. Additionally, prior studies that examined the interplay between human pluripotent stem cells (iPSC and ESC) and ECM elasticity have demonstrated defined substrate stiffness induces stem cell differentiation and lineage specification. In addition, these studies have indicated ECM stiffness provides biomechanical cues for CM functional maturation. HiPSC directed cardiogenesis protocols have improved since their inception, but generating pure and functionally mature populations of hiPSCderived CMs remains a prominent issue. Based on these findings, the ECM has a necessary presence that is absent in feeder-free hiPSC-derived CM cultures. The primary goal of the Lieu laboratory is to investigate the differentiation, enrichment and maturation of hiPSC-derived derived pacemaking and contractile CMs. As a way to contribute to this goal, we examined how the ECM influences CM subtype specification and phenotype maintenance by evaluating properties of the ECM independently to determine the mechanisms by which the ECM niche facilitates differentiation and CM lineage specification into pacemaking and contractile subtypes. We hypothesized that the biochemical and biomechanical properties of the ECM could promote CM subtype specification and facilitate individual functional phenotype maintenance. Our study was organized in two specific aims. The first aim was to determine the reprogramming effects of the ECM microenvironment on hiPSC-derived CM subtype plasticity by performing immunocytochemical (ICC) staining of hiPSC-derived CM markers to quantify protein expression and optical recording of hiPSC-derived action potentials in vitro. The second aim was to determine the effects of the ECM on hiPSC-derived cardiac progenitor cell (CPC) differentiation into contractile and pacemaking CM subtypes by performing immunocytochemical (ICC) staining of hiPSC-derived CM markers in vitro to quantify protein expression. Here, we demonstrated that the expression of pacemaking, contractile, and integrin-binding markers were dependent on different variables of the ECM during hiPSC-derived CM reprogramming and hiPSC-derived CPC differentiation. Furthermore, the electrophysiological properties and subtype distribution of hiPSC- derived CMs were dependent on the unique combination of ECM protein coating and elasticity of the ECM.

This volume provides methodologies for ES and iPS cell technology on the study of cardiovascular diseases. Chapters guide readers through protocols on cardiomyocyte generation from pluripotent stem cells, physiological measurements, bioinformatic analysis, gene editing technology, and cell transplantation studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Pluripotent Stem-Cell Derived Cardiomyocytes aims to help researchers set up experiments using pluripotent stem cell-derived cardiac cells.

Cardiac tissue engineering enables the generation of functional human cardiac tissue using cells in combination with biocompatible materials. Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hPSC-derived cardiac tissues. In the first part of the study, cardiomyocytes derived from human induced pluripotent stem cells (hIPSCs) were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subject to different stress and electrical pacing conditions. This engineered human myocardium exhibits Frank-Starling curve-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated at least 10-fold increase in contractility and tensile stiffness, greater cell alignment, and a 1.5-fold increase in cell size and cell volume fraction within the constructs. Stress conditioning also increased sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA2) expression. When electrical pacing was combined with static stress conditioning, the tissues showed an additional 2-fold increase in force production, tensile stiffness, and contractility, with no change in cell alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. These studies demonstrate that electrical pacing and mechanical stimulation promote both the structural and functional maturation of hiPSC-derived cardiac tissues. In the second part of the study, cardiovascular progenitor (CVP) cells derived from hPSC were used as the input cell population to generate engineered tissues. The effects of a 3-D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue were evaluated. Compared to 2-D culture, the unstressed 3-D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3-D culture suppressed smooth muscle cell maturation. Mechanical stress conditioning further improved cardiomyocyte maturation. Cyclic stress-conditioning increased expression of several cardiac markers, like beta-myosin and cTnT, and the tissue showed enhanced force production. This 3-D system has facilitated understanding of the effect of mechanical stress on the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissues. In conclusion, we were able to create a complex engineered human cardiac tissue with both stem cell-derived cardiomyocytes and CVP cells. We showed that how environmental stimulations like mechanical stress, electrical pacing, and 3-D culturing can affect the maturation and specification of cells within the engineered cardiac tissues. The study paves our way to further apply these engineered cardiac tissues to other in vitro and in vivo usages like drug testing, clinical translation, and disease modeling.

There is great potential for human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) to serve as a test bed for developmental, pharmacological, and regenerative studies. These cells can serve as therapeutic agents, which can be implanted into damage heart tissue to supplant dead cells. They can be used to assess new pharmacological treatments for heart disease. Moreover, they can be used as model systems to study the progression of developmental and pathological states of the heart. However, upon differentiation into cardiomyocytes, these cells are distinctly immature i.e. their cell size, shape, cardiac-specific markers, ploidy, nucleation, calcium handling properties, action potentials, contractility, metabolism, etc. more closely mimic that of an embryonic-stage cardiomyocyte. Therefore, in order for these cells to serve as a valid replacement or model for more developed cardiomyocytes, their structural and functional maturation must be assessed and enhanced. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. Therefore, having the ability to quantify this contraction would provide a powerful assessment tool for hPSC-CMs. Arrays of micropost have previously been employed as a means to measure the isotonic contraction of cardiomyocytes. In this work, a new micropost technique was developed in order to allow for real-time measurements of hPSC-CM contractility, to enable contractile assessment under various different culture conditions. Previous studies with immature cardiomyocytes have shown that a number of different methods are able to enhance their contractile and structural maturation. Here, hPSC-CM maturation was achieved via: i) prolonged cell culture, ii) cell alignment, iii) controlling cell-cell contact between adjacent cells, iv) altering substrate stiffness, v) electrically-stimulating the cells, and vii) treating the cells with various different biochemical agents. Assessment of hPSC-CM structural maturation was achieved by immunofluorescent analysis, while high speed imaging of micropost deflections and fluorescent calcium transients was used to quantify functional maturation. Through these studies, I found that the micropost assay is capable of accessing the contractile state of immature human cardiomyocytes, which makes it a powerful tool for developmental studies, pharmacological screening, and disease modeling applications. Furthermore, the pro-maturation environment that I developed was able to elicit cardiomyocyte maturation in the absence of any biochemical cues. Ultimately, I believe that these novel culture and analysis techniques will provide future researchers with a means to culture large populations of rapidly matured stem cell-derived cardiomyocytes, in order to effectively perform developmental, pharmacological, and therapeutic studies in a more rapid and high-throughput manner.

This volume provides methodologies for ES and iPS cell technology on the study of cardiovascular diseases. Chapters guide readers through protocols on cardiomyocyte generation from pluripotent stem cells, physiological measurements, bioinformatic analysis, gene editing technology, and cell transplantation studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Pluripotent Stem-Cell Derived Cardiomyocytes aims to help researchers set up experiments using pluripotent stem cell-derived cardiac cells.

This book discusses critical areas of progress in stem cell research, including the most recent research and applications of pluripotent embryonic cells, induced pluripotent cells, oligopotent tissue stem cells and cancer stem cells. The text covers basic knowledge of stem cell biology, stem cell ethics, development of techniques for applying stem cell therapy, the technology of obtaining appropriate cells for transplantation as well as the role of stem cells in cancer and how therapy may be directed to cancer stem cells. This new volume is essential reading for all scientists currently in the field or allied research areas, and those for those graduate students who envision a career in stem cells.