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.

Heart diseases remain the leading cause of morbidity and mortality worldwide. As damages done to the heart are irreversible, heart transplant is the ultimate therapy, but it is greatly limited by the shortage of heart donors. Thus, scientists are attracted by induced pluripotent stem cells (iPSCs) as a solution because of their ability to be reprogrammed from a somatic cell source, potentially unlimited proliferative properties, and ability to be differentiated into many different cell types. However, hiPSC-derived cardiomyocytes display immature phenotypes in contractile properties, electrophysiology, metabolism, structure, and protein isoform expression, thus greatly limiting their application in regenerative medicine, disease modeling, and drug screening. Therefore, there is a great need for a technique to drive the maturation of stem cell-derived cardiomyocytes to better recapitulate the properties of their adult counterpart. Our approach was to recreate a developmentally-inspired microenvironment for maturing hiPSC-derived cardiomyocytes (hPSC-CMs). The native myocardium is characterized by aligned extracellular matrix (ECM) fibers and cells have been shown to sense and respond to cues in the ECM. In addition, thyroid hormone is a major regulator of heart development in promoting cell hypertrophy and elongation. Thus, we tested the effects of biomimetic, nanotopographical cues – using an anisotropic nanofabricated substrata (ANFS) composed of nanogrooves and nanoridges in the nanopattern (NP) – combined with thyroid hormone T3 on the structural maturation of cardiomyocytes. We found that cells exposed to nanotopography exhibited structural organization and maturation. However, the effect of T3 was not clear and appeared to have a detrimental effect at prolonged exposure at high concentration. ANFS was also used to differentiate cardiomyocytes from the cardiac progenitor stage and suggested nanotopography could have a positive effect on cardiomyocyte differentiation yield. However, experiments suggested that the differentiating cell population was highly dynamic and responded differently to the replating procedure at different time points. Therefore, a photothermal-responsive polymer was developed to introduce nanotopography with an external light stimulus, and cells were confirmed to stay attached to the polymer substrate with the topographical switch. This resulted in the development of an effective platform with vast potential, allowing the introduction of topographical cues to a cell culture with an easily manipulated external stimulus.

To achieve cardiac regeneration using pluripotent stem (iPS) cells, researchers must understand iPS cell generation methods, cardiomyocyte differentiation protocols, cardiomyocyte characterization methods, and tissue engineering. This book presents the current status and future possibilities in cardiac regeneration using iPS cells. Written by top researchers who present new data in these fields, this book reviews cardiac cell therapy for ischemic heart disease and explores in vitro generation of efficacious platelets from iPS cells. It also discusses modeling arrhythmogenic heart disease with patient-specific induced pluripotent stem cells.

Stem cell and regenerative medicine research is a hot area of research which promises to change the face of medicine as it will be practiced in the years to come. Challenges in the 21st century to combat diseases such as cancer, Alzheimer and related diseases may well be addressed employing stem cell therapies and tissue regeneration. Frontiers in Stem Cell and Regenerative Medicine Research is essential reading for researchers seeking updates in stem cell therapeutics and regenerative medicine. The seventh volume of this series features reviews on roles of mesenchymal stem cells and biomaterials in cartilage regeneration in vivo, liver regeneration, cardiogenesis and magnetic nanoparticles for regenerative therapy.

Calcium is crucial in governing contractile activities of myofilaments in cardiomyocytes, any defeats in calcium homeostasis of the cells would adversely affect heart pumping action. The characterization of calcium handling properties in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMCs) is of significant interest and pertinent to the stem cell and cardiac regenerative field because of their potential patient-specific therapeutic use.

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.