Browsing by Subject "Cardiomyocyte"

Now showing 1 - 4 of 4
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    Characterization of Maturity Marker Cardiac Troponin I in Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes
    (2018-12) Pelaez, Andres
    The use of human-induced pluripotent stem cells (hiPSCs) have opened new possibilities when it comes to medical discovery and patient care. One avenue that has benefitted is the cardiovascular field. Attempts to increase patient life after a cardiovascular event have led to increasingly better results as time has gone on. Yet, one issue that arises is the maturity level of hiPSC-derived cardiac myocytes (hiPSC-CMs). Current guidelines state the importance of being 100% certain that the cell type in question has fully differentiated into another, meaning that they should contain the same phenotype, markers and function as the wildtype cells in question. Cardiac myocytes have thus been difficult to consider mature due to the lack of certain maturity markers available. In this study, the irreversible, stoichiometric isoform switch between cardiac troponin I (cTnI) and slow-skeletal troponin I (ssTnI) is used to increase cTnI expression levels in the hiPSC-CMs. Promising results were found in all cases, with the adenovirus transduction of cTnI and the transfection of antisense oligonucleotides (ASOs) yielding increased or decreased cTnI expression in hiPSC-CMs. This lays the framework for further experimentation to analyze cardiac myocyte maturity via the cTnI marker.
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    Enhancing selection and biological applications of oligonucleotide affinity reagents
    (2021-08) Dembowski, Sean
    Aptamers are unique sequences of single-stranded DNA or RNA that exhibitsignificant affinity toward a particular target of interest. They have a number of advantages over antibodies – ease of modification, cell-free synthesis, and spontaneous renaturation – yet the preference of most researchers for antibodies continues due to prevalence of commercial antibody sources, hesitance adjusting existing research protocols, and a subconscious association of binding behavior with proteins rather than nucleic acids. While aptamers have already shown their technical advantages in the literature, they will also need to demonstrate their practicality via a broader range of targets and useful applications in order for aptamers to become more widespread in bioanalytical research. One challenge aptamers have faced is the inconsistency of SELEX, the combinatorial method by which new aptamers are identified, as well as a small pool of potential aptamer targets. This dissertation demonstrates the feasibility of aptamer selection against intact membrane proteins using a capillary electrophoresis-based SELEX technique, an achievement not yet demonstrated in the literature that opens SELEX to a much wider range of biologically significant potential targets. In addition to high affinity binding (Kd < 10 nM), these aptamers should undergo cellular internalization with their membrane receptor target, allowing hybrid aptamers to reach previously inaccessible intracellular targets as well. Additionally, this dissertation serves to increase the body of work demonstrating the bioanalytical capabilities of aptamers and other oligonucleotide affinity reagents. First, progress is shown toward aptamers targeting mouse leptin for use in a real-time aptamer- based microfluidic assay to quantify leptin secretion in cultured adipocytes. This proposed device would allow measurement of leptin, an important metabolic protein related to obesity, on a sub-minute time scale in response to metabolic stimuli. Quantification involves on-line separation of aptamers and aptamer-leptin complexes by electrophoresis, a technique that lends itself well to oligonucleotide affinity reagents due to their highly negative charge. Finally, a heart model based on cardiomyocyte differentiation from induced stem cells is demonstrated in preparation for measurement of physiological effects of aptamer-like SPIDRs (small protein-interacting DNAs and RNAs) on cardiomyocyte beating. SPIDRs have been shown to bind to the calcium pump SERCA and its control protein phospholamban, increasing the calcium affinity of SERCA. The cardiomyocyte model will be used to evaluate the hypothesis that this increase in calcium affinity leads to measurable physiological changes in beating, namely the beat rate and fall time, demonstrating the therapeutic potential of SPIDRs. Herein, optimization of stem cell growth and differentiation to cardiomyocytes are shown, as well as preliminary quantitative data regarding the reproducibility of cell impedance measurements.
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    Human iPSC-Derived Cardiac Myocytes: Toward an In Vitro Model of Cardiac Physiology
    (2017-05) Wheelwright, Matthew
    Cardiovascular Disease is a growing public health issue in the modern world, with a high incidence rate that continues to increase, and poor mortality rates. Recent technological advances have made it possible to efficiently derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). These have been seen as a model for human heart disease, as well as a potential source for cellular transplantation into failing diseased heart tissue. Many laboratories have devoted substantial effort to examining the functional properties of hiPSC-CMs, including electrophysiology, intracellular calcium handling, and gene/protein expression and force. In the first part of this thesis, we utilize traction force microscopy (TFM) to determine the maximum force production of isolated hiPSC-CMs under varied culture and assay conditions. We elucidate here the relationship between cell morphology and force production, and find a significant relationship between cell size and force. HiPSC-CMs developing in culture for two weeks produce significantly less force than cells cultured from one to three months and hiPSC-CMs cultured for three months resemble the cell morphology of neonatal rat ventricular myocytes. Unexpectedly, hiPSC-CMs produce less force when assayed on increasingly stiff substrates, and generate less strain energy. Finally, hiPSC-CMs cultured in conditions of physiologic calcium concentrations are larger and produce more force than cells cultured in standard media. In the second part of this thesis, we address the concept of immaturity in hiPSC-CMs, and attempt to accelerate maturation. We use genome editing to engineer hiPSC-CMs that contain an inducible gene expression cassette, in order to overexpress two proteins associated with maturity: SERCA2a and cardiac troponin I (cTnI). We find that we are able to overexpress both proteins in differentiated hiPSC-CMs after two weeks of treatment with doxycycline. SERCA2a-overexpressing cells showed significant alterations in physiologic function, including increased chronotropy and decreased time to peak in calcium transients following treatment with isoproterenol, a β-adrenergic agonist. Furthermore, using an impedance-measuring system to track contractility kinetics, we found that SERCA2a-overexpressing cells had shortened time to peak and time to baseline after gene induction, with continued response to isoproterenol. As a sign of maturation, SERCA cells also expressed increased cTnI, a key marker of maturity. Using RNAseq, we found that cTnI-overexpressing cells had marked, global changes in their gene expression profile. Key findings include upregulation of genes associated with cardiac contractility and development, such as cardiac myomesin and tropomyosin and ryanodine receptor, and downregulation of genes associated with pacemaker and ventricular cell types, such as HCN and GREM2, and genes associated with skeletal myocytes, such as skeletal muscle actin. Overall, our findings show that hiPSC-CMs have physiologic function similar to that of immature cardiac myocytes, but that we are able to induce maturation by overexpression of genes associated with maturity.
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    Myocardial Endoglin Regulates Cardiomyocyte Proliferation and Cardiac Regeneration
    (2022-03) Sorensen, Daniel
    The mammalian heart loses almost all its regenerative potential in the first week of life due to the cessation of the ability of cardiomyocytes to proliferate. In recent years, a number of regulators of cardiomyocyte proliferation have been identified. Despite this, a clear understanding of the regulatory pathways that control cardiomyocyte proliferation and cardiac regeneration is lacking, and there are likely additional regulators to be discovered. Here, we performed a genome-wide screen on fetal murine cardiomyocytes to identify potential novel regulators of cardiomyocyte proliferation. Endoglin was identified as an inhibitor of cardiomyocyte proliferation in vitro. Endoglin knock-down resulted in enhanced DNA synthesis, cardiomyocyte mitosis and cytokinesis in mouse, rat and human cardiomyocytes. Using gene-targeted mice, we confirmed myocardial Endoglin to be important in cardiomyocyte proliferation and cardiac. Mechanistically, we show that Smad signaling is required for the endoglin-mediated anti-proliferative effects. Our results identify the TGF-β coreceptor Endoglin as a regulator of cardiac regeneration and cardiomyocyte proliferation.