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.

Cardiovascular disease continues to be a leading cause of death worldwide motivating the need for models of cardiac function in both healthy and pathological conditions for basic science and clinically translational research. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells provide a source for developing in vitro cardiac models, however current in vitro culture strategies and analysis techniques provide only a portion of the necessary means to fully characterize functionality. Specifically, there is a need for additional techniques to quantify and understand the contractile behavior of CMs as they interact collectively with each other and their surroundings. The following chapters describe the utility of an engineered culture platform that enables full field mechanical analysis resulting in new findings regarding CM behavior in healthy and disease models. Using a tailorable, biologically relevant platform, the influence of substrate properties and extracellular matrix (ECM) characteristics are explored. Digital Image Correlation (DIC) is used, and additional mechanical analysis tools were developed to extend the utility of the analysis pipeline. The platform was used to examine interactions between cardiac fibroblasts (CFs) and CMs, as well as the interactions of these cells with the ECM. iPSC-CFs remodel and produce aligned ECM as well as increasing contractile strain when in co-culture with iPSC-CMs. When seeded on decellularized ECM, the contractile strain was found to be higher for iPSC-CMs in co-culture with iPSC-CFs, but there was no significant difference between ECM conditions. iPSC-CMs maintained spatial organization of their contractions in co-culture with iPSC-CFs. The functionality of this platform as a disease model was then demonstrated, first as a model of catecholaminergic polymorphic ventricular tachycardia (CPVT) using iPSC-CMs from a CPVT patient (RyR2-H2464D mutation) and a healthy familial control. The maximum contractile strain was found to be consistently higher in iPSC-CMs derived from the patient compared to the familial control across three different substrate stiffnesses. Additionally, the patient cell line had a statistically significantly slower intrinsic contraction rate than the control. A hypertrophic cardiomyopathy (HCM) disease model was created using CRISPR/Cas9 modified iPSCs deficient in the cMyBP-C. The model recapitulated the increased contractile function of CMs with a homozygous knockout prior to hypertrophic remodeling. The utility of the platform was further demonstrated by evaluating the response to stimuli such as substrate stiffness and patterned features and analyzing contractile kinetics. These combined results highlight the utility of the platform as an in vitro cardiovascular model and will allow for further understanding of the interplay of genetics, environment, and genotype-phenotype relationships.

This dissertation, "Calcium Signaling in Human Pluripotent Stem Cell-derived Ventricular Cardiomyocytes" by Sen, Li, 李森, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs) for disease modeling, cardiotoxicity screening, drug discovery and cell‐based therapies. However, as shown in previous studies conducted by our lab (Poon, Kong et al. 2011), human embryonic stem cells (hESCs)‐derived CMs display immature〖Ca〗 DEGREES(2+)-handing properties with smaller transient amplitudes, slower rise and decay kinetics than those of adult CMs. Although the cytosolic 〖Ca〗 DEGREES(2+) signaling of hESC‐CMs has only recently been understood, there is no investigation on the nuclear 〖Ca〗 DEGREES(2+) signal in hESC‐CMs, despite its importance. In this dissertation, delayed kinetics of nuclear 〖Ca〗 DEGREES(2+), as compared to that of cytosol during 〖Ca〗 DEGREES(2+)waves or 〖Ca〗 DEGREES(2+) transients, was found in hESC‐derived ventricular (V) CMs, indicating that nuclear 〖Ca〗 DEGREES(2+) was initiated by 〖Ca〗 DEGREES(2+) diffusion from cytosol. Besides global 〖Ca〗 DEGREES(2+) signals, local nuclear 〖Ca〗 DEGREES(2+) signals were observed and identified as Ca2+ release from ryanodine receptors (RyRs), and nucleoplasmic reticulum (NR) served as their structural basis. In addition, targeted expression of 〖Ca〗 DEGREES(2+) buffering protein parvalbumin (PV) in cytosol or nucleus altered 〖Ca〗 DEGREES(2+) transient and stimuli‐induced apoptosis of hESC‐VCMs. For cytosolic 〖Ca〗 DEGREES(2+) signaling in hESC‐VCMs, the mechanistic basis of excitation‐contraction coupling of hESC‐VCMs was studied by using 〖Ca〗 DEGREES(2+) sparks, which are the unitary 〖Ca〗 DEGREES(2+) ‐events. The results indicated that RyRs could be sensitized by 〖Ca〗 DEGREES(2+) in permeabilized hESC‐VCMs. Increasing external 〖Ca〗 DEGREES(2+) dramatically escalated the basal 〖Ca〗 DEGREES(2+) and spark frequency. Furthermore, RyR‐mediated Ca2+ release sensitized nearby RyRs, leading to compound 〖Ca〗 DEGREES(2+) sparks, whereas inhibition of mitochondrial 〖Ca〗 DEGREES(2+) + uptake promoted Ca2+ waves. The aforementioned immature 〖Ca〗 DEGREES(2+)-handing properties of hESC‐CMs can be attributed to their differential expression of crucial Ca2+-handling proteins. During diastole, SERCA and NCX sequester and extrude 〖Ca〗 DEGREES(2+) ions, respectively, to return cytosolic 〖Ca〗 DEGREES(2+) to the resting level. As previously published in our lab, NCX, robustly expressed in hESC‐CMs but much less so in the adult counterparts, is a functional determinant of immature 〖Ca〗 DEGREES(2+) homeostasis. Unlike NCX, SERCA is expressed less in hESC‐CMs than in adult‐CMs. The present study first demonstrated the effects of lentivirus‐based genetic manipulation of SERCA2a and NCX1 in hESC‐VCMs, and the results indicated that SERCA2a overexpression shortened the decay phase of low‐frequency (0.5 Hz) electrical stimulation‐elicited Ca2+ transient. Increasing pacing frequency from 0.5 Hz to 2 Hz led to a decrease of relative transient amplitude, showing that hESC‐VCMs harbored a negative‐frequency response. At a high‐stimulation frequency of 2 Hz, it was revealed that SERCA overexpression, but not NCX1 suppression, increased the amplitude of 〖Ca〗 DEGREES(2+) transient by accelerating 〖Ca〗 DEGREES(2+) sequestration to sarcoplasmic reticulum (SR), indicating partial rescue of the negative‐frequency response. T

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.