Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.
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Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.
Published Version (Please cite this version)
Pomeroy, Jordan E, Hung X Nguyen, Brenton D Hoffman and Nenad Bursac (2017). Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells. Theranostics, 7(14). pp. 3539–3558. 10.7150/thno.20593 Retrieved from https://hdl.handle.net/10161/15568.
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The overall goal of my research program is to utilize an interdisciplinary approach to first advance the basic understanding of mechanotransduction on multiple scales and then use this knowledge to guide the development of new treatments for mechanosensitive diseases. Our work combines principles and techniques from protein engineering, molecular biology, soft matter physics, cell and developmental biology, biomaterials engineering, automated image analysis, and state of the art live cell microscopy. Specifically, we engineer and use biosensors that report the tension across specific proteins in living cells through changes in the color of light they emit. This technology enables dynamic measurements of proteins and sub-cellular structures that are under load. Unlike more traditional techniques that measure the entirety of cellular force output, the ability of these sensors to measure mechanical stress at the molecular level means they are innately compatible with concepts and approaches common in molecular biology and biophysics.
Bursac's research interests include: Stem cell, tissue engineering, and gene based therapies for heart and muscle regeneration; Cardiac electrophysiology and arrhythmias; Organ-on-chip and tissue engineering technologies for disease modeling and therapeutic screening; Small and large animal models of heart and muscle injury, disease, and regeneration.
The focus of my research is on application of pluripotent stem cells, tissue engineering, and gene therapy technologies for: 1) basic studies of striated muscle biology and disease in vitro and 2) regenerative therapies in small and large animal models in vivo. For in vitro studies, micropatterning of extracellular matrix proteins or protein hydrogels and 3D cell culture are used to engineer rodent and human striated muscle tissues that replicate the structure-function relationships present in healthy and diseased muscles. We use these models to separate and systematically study the roles of structural and genetic factors that contribute cardiac and skeletal muscle function and disease at multiple organizational levels, from single cells to tissues. Combining cardiac and skeletal muscle cells with primary or iPSC-derived non-muscle cells (endothelial cells, smooth muscle cells, immune system cells, neurons) allows us to generate more realistic models of healthy and diseased human tissues and utilize them to mechanistically study molecular and cellular processes of tissue injury, vascularization, innervation, electromechanical integration, fibrosis, and functional repair. Currently, in vitro models of Duchenne Muscular Dystrophy, Pompe disease, dyspherlinopathies, and various cardiomyopathies are studied in the lab. For in vivo studies, we employ rodent models of volumetric skeletal muscle loss, cardiotoxin and BaCl2 injury as well as myocardial infarction and transverse aortic constriction to study how cell, tissue engineering, and gene (viral) therapies can lead to safe and efficient tissue repair and regeneration. In large animal (porcine) models of myocardial injury and arrhythmias, we are exploring how human iPSC derived heart tissue patches and application of engineered ion channels can improve cardiac function and prevent heart failure or sudden cardiac death.
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