Browsing by Subject "Disease modeling"
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Item Open Access 3D Human Skeletal Muscle Model for Studying Satellite Cell Quiescence and Pompe disease(2021) Wang, JasonTissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.
Item Open Access Tick-borne Disease Risk along the Appalachian Trail(2012-04-25) Shelus, VictoriaEach year, 2-3 million visitors walk a portion of the Appalachian Trail, engaged in outdoor activities where exposure to ticks is likely. While the trail passes through the states with the greatest number of cases of Lyme disease and Rocky Mountain Spotted Fever, it is unknown how many people become sick after visiting the trail. This paper assesses tick-borne disease risk in the National Park Service (NPS) units located along the Appalachian Trail, and finds that the disease risk is unknown, and likely under recognized. It is recommended that tick sampling as part of a larger tick-borne disease surveillance program be implemented in the national parks. As a starting point to further study, general tick habitat suitability was modeled for the NPS units along the Appalachian Trail based on land cover, elevation and moisture. Potential tick sampling sites were selected based on areas of high tick habitat suitability and high visitor use.Item Open Access Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome(2022) Abutaleb, Nadia OsamaHutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal genetic disease that causes progressive atherosclerosis and accelerated aging in children resulting in fatality at an average of 14.6 years of age. With a limited pool of HGPS patients, clinical trials face unique challenges and require reliable preclinical testing. Preclinical studies to date have relied on 2D cell culture using HGPS fibroblasts which does not accurately model the 3D physiological microenvironment and limits the scope many studies to only the fibroblast response, which may not translate to a vascular benefit. Further, only two HGPS murine models develop atherosclerosis and these exhibit symptoms that are not present in HGPS patients. An ideal model would incorporate human cells in a microenvironment mimicking in vivo conditions and replicate key aspects HGPS vascular pathology. Such a model would contribute significantly to the ability to study HGPS mechanisms and test therapies. One significant area of research that requires further study is the contribution of endothelial cells to HGPS pathology. The endothelium plays a critical atheroprotective role in maintaining vascular homeostasis. When it is damaged, dysfunctional endothelium becomes inflammatory and proatherogenic, contributing significantly to atherosclerosis and cardiovascular disease. Progressive atherosclerosis is the most severe symptom of HGPS and is the common underlying cause of mortality in HGPS patients, yet only a few studies have investigated the HGPS endothelial phenotype. The goal of our work was to develop and characterize a tissue engineered model of HGPS vasculature that could be used to study endothelial dysfunction in HGPS and test therapies to alleviate HGPS vascular pathology.In Aim 1, we developed and characterized a tissue engineered blood vessel (TEBV) model of HGPS using vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) differentiated from induced pluripotent stem cells (iPSCs) from two HGPS patients. HGPS viSMCs and viECs exhibited manifestations consistent with typical symptoms of HGPS including progerin expression, abnormal nuclear morphology, and premature senescence. HGPS viECs exhibited cell responses consistent with endothelial dysfunction including impaired tube formation, elevated reactive oxygen species (ROS) levels, reduced proliferation, and increased levels of DNA damage compared with healthy viECs. HGPS viECs also displayed a blunted response to shear stress including limited flow-sensitive gene expression and reduced nitric oxide production. TEBVs fabricated with HGPS cells exhibited reduced vasoactivity compared with healthy TEBVs and replicated HGPS vascular pathology including SMC loss, excess extracellular matrix (ECM) protein deposition, inflammation, and vascular calcification. In Aim 2, we tested the effects of HGPS therapeutics the farnesyltransferase inhibitor Lonafarnib and mTOR inhibitor Everolimus, currently in Phase I/II clinical trial, on endothelial dysfunction and HGPS TEBVs phenotype. Everolimus decreased reactive oxygen species levels, increased proliferation, and reduced DNA damage in HGPS vascular cells. Lonafarnib improved flow-sensitive gene expression of HGPS viECs exposed to physiological shear stress. Both Lonafarnib and Everolimus were able to restore nitric oxide production to healthy levels in HGPS viECs exposed to physiological shear stress. While Everolimus improved vasoconstriction, Lonafarnib increased vasodilation, ECM deposition, inflammation, and calcification in HGPS TEBVs. Combination treatment with Lonafarnib and Everolimus maintained the benefits of each monotherapy and also resulted in additional benefits such as improved endothelial marker expression and reduced apoptosis. The 3D TEBV model was critical to reveal the benefit of Lonafarnib and Everolimus combination treatment which was more limited in 2D studies but became clear in the TEBVs. In Aim 3, we tested a recently developed targeted gene therapy for HGPS known as adenine base editing (ABE). ABE corrects the genetic heterozygous point mutation that causes HGPS with high efficiency and minimal off-target effects. ABE ameliorated all the dysfunctional endothelial phenotypes that we tested in HGPS viECs including elevated ROS levels, reduced proliferation, and increased DNA damage. Most critically for endothelial function, ABE restored nitric oxide production and flow-sensitive gene expression to healthy levels in HGPS viECs. In TEBVs, ABE restored healthy levels of vasoconstriction and vasodilation, improved SMC retention, increased proliferation, and inhibited excess ECM protein expression. In summary, this work contributes data supporting the hypothesis that progerin induces endothelial dysfunction in HGPS endothelial cells which could contribute to the vascular pathology observed in HGPS. This work also tests current and novel therapies in the first 3D tissue engineered model of HGPS, validating the model as a valuable platform for preclinical testing that can supplement and improve information gathering from current 2D and animal models.