1,720,989 research outputs found
Characterization of LINC Complex-mediated Regulation of Satellite Stem and Progenitor Cell Cycles
Full text linkEmery-Dreifuss muscular dystrophy (EDMD) is an inherited disorder characterized by skeletal muscle wasting and cardiomyopathy. EDMD is linked to mutations affecting proteins forming the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex, composed of Nesprins at the outer nuclear membrane interacting with SUNs at the inner nuclear membrane, creates a bridge linking the cytoskeleton and nuclear lamina. The role of the LINC complex in the regulation of satellite stem and progenitor cell cycles is currently unknown. Using dominant-negative and siRNA approaches, we find that disrupting the LINC complex in satellite cells (SC) reduces regenerative potential and SC self-renewal in-vivo. Furthermore, we find that LINC complex disruption alters myoblast cell cycle. Our results provide evidence that EDMD may be due in part to defective SC function. Future studies aim to uncover molecular mechanisms through which the LINC complex modulates myogenic cell cycle to identify therapeutic entry-points for EDMD.M.Sc
Development of a 96-well Platform for the Bulk Production of Three-Dimensional Human Skeletal Muscle Microtissues
No full textSkeletal muscle wasting is a largely untreatable condition wherein patients lose skeletal muscle strength, stamina, and protein content. We posit that the lack of skeletal muscle wasting treatments is due to the failings of existing skeletal muscle models. To address this problem, we developed a human muscle microtissue (hMMT) platform for bulk generation of reproducible, contractile, 3D hMMTs. hMMTs recapitulated key aspects of native human skeletal muscle, including muscle-specific protein expression, drug response, and contractility. Furthermore, the hMMT platform can non-invasively assess strength, and is exceedingly simple to fabricate. To illustrate hMMT platform utility, hMMTs were used with tumour-conditioned media to model cancer-associated muscle wasting in vitro. hMMTs were also used to screen chemotherapeutics, revealing that the drug irinotecan is toxic to human myofibers. Collectively, these results underline the considerable potential of the hMMT platform as a vehicle for skeletal muscle research and drug candidate validation.M.A.S
The Role of Niche Stiffness on Muscle Stem Cell Symmetric Self-renewal
Full text linkReconstruction of the adult muscle tissue relies on stem cells situated in a niche between the basal lamina and sarcolemma, but the molecular mechanisms of cell fate decisions, especially self-renewal, during muscle regeneration remains unclear. We report that the local extracellular matrix surrounding the muscle stem cell in healthy, uninjured tissue is permissive for planar and apical-basal division orientations that lead to either symmetric or asymmetric cell fates, but that the increased extracellular matrix deposition during injury physically alters the niche architecture to significantly favour symmetric divisions in the planar orientation. We demonstrate that the addition of soluble ligands such as Wnt7a leads to a significant increase of self-renewal divisions instead of differentiation, but only when coupled with 3D culture in a stiff environment. Together, these results suggest that biophysical cues such as niche stiffness work synergistically with biochemical cues like Wnt7a signaling to regulate muscle stem cell self-renewal.M.A.S
A Multi-angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies
Full text linkSkeletal muscle plays an essential role in locomotion, metabolism, and thermoregulation. An intriguing characteristic of skeletal muscle is its remarkable regenerative capacity: a highly orchestrated cellular process, in which the resident muscle stem cells (MuSCs) play a central role. Upon tissue injury, quiescent MuSCs are activated and give rise to a population of primary myoblasts (pMBs). pMBs undergo several rounds of division and ultimately fuse with one another to form multi-nucleated myofibers and repair the muscle. As observed in a wide range of conditions such as muscular dystrophies and aging, disruptions in the repair process can lend to impaired regeneration and progressive muscle wasting. Strategies to restore strength and function to pathological muscle include cell-based therapies to replace defective myogenic cells, and treatments to restore the endogenous repair process. Despite substantial advances, these treatment options are still in the early stages of clinical translation. The specific goal of this thesis is to improve upon currently available strategies as well as to lay the groundwork for the emergence of new therapeutic entry-points for skeletal muscle treatments.
In the area of cell transplantation therapy, challenges remain in producing clinically-relevant numbers of cells that, as a population, possess high regenerative potency to produce skeletal muscle and repopulate the stem cell niche. We demonstrate that by using a bioactive hydrogel (HAMC) as the cell delivery vehicle, it is possible to improve MuSC transplantation outcomes, and thereby reduce the overall number of required MuSCs. Next, we address the issue of producing sufficient numbers of highly regenerative myogenic cells. Using a high-throughput drug screen and in-vivo intramuscular transplantation assay validation, we identify epidermal growth factor receptor (Egfr) and vascular endothelial growth factor receptor 2 (Vegfr2, Kdr) as new druggable targets, that upon inhibition, produce a population of cultured MuSCs with greater regenerative potency than control treated. Finally, using single-cell RNA sequencing, we shed light on the diversity and intercommunication of cells present in skeletal muscle. This dataset serves as a valuable resource through which new regulators of MuSCs and other cells in skeletal muscle can be evaluated with an eye towards skeletal muscle regenerative medicine applications.Ph.D
Investigating Denervation Atrophy Through Prolonged Culture of Engineered Skeletal Muscle Tissues: The Good, the Bad, and the Ugly
No full textModern advancements in 3D skeletal muscle tissue engineering have provided an opportunity to develop a cell-based denervation culture model. The opening approach to establish denervation atrophy in vitro was through the prolonged culture of human immortalized 3D engineered skeletal muscle tissues. Myotube diameter, sarcomere organization, and calcium handling properties were assessed across a wide range of experimental endpoints to establish a purely myogenic tissue healthspan. A pattern of increasing muscle maturation for the first two weeks, then a slow decrease due to deterioration was observed. While evidence of denervation atrophy was detected, evidence of biomechanical instability was also implicated. Additionally, tissue variability and failure hindered results and prevented well drawn conclusions. Fibroblast co-cultures were constructed to improve tissue consistency, but experiments were unsuccessful. Thus, while this model holds promise, tissue variability and failure need to be reduced before further development and validation can be achieved.M.A.S
Engineered In Vitro Models of Human Skeletal Muscle for Drug Testing, Developmental Biology, and Physiology Studies
No full textSkeletal muscle is the most abundant tissue in the lean human body and is responsible for all motor functions such as locomotion, speech, and respiration. During development, myogenic progenitor cells fuse to form multinucleated muscle fibers that are innervated by the motor neurons of the spinal cord to form functional motor units. Various genetic and acquired diseases of motor neuron, neuromuscular junction (NMJ), and skeletal muscle affect motor unit function with significant effects on patients’ quality of life. Recent advancements in the field of tissue engineering now enable precise control of microenvironment and biochemical cues that emulate physiological conditions and have led to the successful formation of functional human skeletal muscle and neuromuscular tissues in vitro. In 2013 when I initiated my Ph.D. studies, the field had yet to establish parameters to produce engineered human skeletal muscle tissue in a dish capable of responding to electrical and biochemical stimuli. Further, human neuromuscular co-culture models to study NMJ transmission and diseases impacting this critical structure were replete. Since 2013, my work and that of other researchers has overcome barriers to forming functional of human-cell derived skeletal muscle and neuromuscular tissues in vitro. The work in this thesis leveraged, extended, and then applied prior advancements in tissue engineering and engineering techniques, in general, to improve our understanding of skeletal muscle, the neuromuscular junction formation and the maturation of each, in vitro. An important goal of my studies was to encourage widespread adoption of methods I developed by researchers and industry. As such, the tools and technologies were engineered to be simple and integrate into current standard operating procedures and the experimental studies were focused on uncovering biological inquiries that were new and/or were not possible to study with other currently available culture or animal models.Ph.D
Investigating ICU-acquired Weakness in a 3D Human Skeletal Muscle Microtissue Platform
No full textNumerous pathologies can lead to progressive muscle weakness and atrophy as secondary consequences. Intensive care unit acquired weakness (ICUAW) is a clinically detected muscle weakness in critically-ill patients, often resulting in persistent disability. We investigated how humoral factors contribute to ICUAW by treating genetically identical 3D-human skeletal muscle microtissues(hMMTs) with sera collected along the treatment of critically-ill patients that may develop ICUAW. Treatment of hMMTs with sera collected within 72 hours of ICU admission with intubation and mechanical ventilation resulted in significantly reduced hMMT myotube diameter, sarcomere integrity and absolute passive force. Comparison of trends in hMMT form and function, when treated with early and later timepoint serum, to clinical outcomes revealed that hMMT passive force outcomes align with clinical outcomes of function measured by Medical Research Council sum score and motor functional independence measure. The next steps will include understanding which molecules are responsible for effects observed in culture.M.A.S.2022-11-30 00:00:0
A Multi-angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies
No full textSkeletal muscle plays an essential role in locomotion, metabolism, and thermoregulation. An intriguing characteristic of skeletal muscle is its remarkable regenerative capacity: a highly orchestrated cellular process, in which the resident muscle stem cells (MuSCs) play a central role. Upon tissue injury, quiescent MuSCs are activated and give rise to a population of primary myoblasts (pMBs). pMBs undergo several rounds of division and ultimately fuse with one another to form multi-nucleated myofibers and repair the muscle. As observed in a wide range of conditions such as muscular dystrophies and aging, disruptions in the repair process can lend to impaired regeneration and progressive muscle wasting. Strategies to restore strength and function to pathological muscle include cell-based therapies to replace defective myogenic cells, and treatments to restore the endogenous repair process. Despite substantial advances, these treatment options are still in the early stages of clinical translation. The specific goal of this thesis is to improve upon currently available strategies as well as to lay the groundwork for the emergence of new therapeutic entry-points for skeletal muscle treatments.
In the area of cell transplantation therapy, challenges remain in producing clinically-relevant numbers of cells that, as a population, possess high regenerative potency to produce skeletal muscle and repopulate the stem cell niche. We demonstrate that by using a bioactive hydrogel (HAMC) as the cell delivery vehicle, it is possible to improve MuSC transplantation outcomes, and thereby reduce the overall number of required MuSCs. Next, we address the issue of producing sufficient numbers of highly regenerative myogenic cells. Using a high-throughput drug screen and in-vivo intramuscular transplantation assay validation, we identify epidermal growth factor receptor (Egfr) and vascular endothelial growth factor receptor 2 (Vegfr2, Kdr) as new druggable targets, that upon inhibition, produce a population of cultured MuSCs with greater regenerative potency than control treated. Finally, using single-cell RNA sequencing, we shed light on the diversity and intercommunication of cells present in skeletal muscle. This dataset serves as a valuable resource through which new regulators of MuSCs and other cells in skeletal muscle can be evaluated with an eye towards skeletal muscle regenerative medicine applications.Ph.D
Adapting Muscle Endogenous Repair (MEndR) Assay for Academic and Industry Adoption
No full textSkeletal muscle is an essential tissue and in healthy individuals it can self-repair through the activity of muscle stem cells (MuSCs). In age and disease, MuSC endogenous repair proficiency is lost, and therapeutics that target and restore functionality are of interest. However, in vivo validation of drugs with potential to stimulate muscle endogenous repair proves to be a bottleneck in drug discovery and to address this problem we developed a MuSC mediated skeletal muscle endogenous repair (MEndR) phenotypic culture assay that predicts gold-standard in vivo assay outcomes. Roadblocks exist that prevent scale-up, and assay adoption. My thesis addresses two issues: phenotypic data analysis and the use of cells with access and scale-up limitations. My thesis offers a semi-automated pipeline to analyze MuSC-derived myotubes from confocal images with the power to stratify MuSC drug treatment response. I also identify immortalized myoblast cell lines with which to engineer the muscle templates.M.A.S.2024-03-24 00:00:0
Positioning Microtissues on the Differentiation Continuum Reveals their Utility for Studying Skeletal Myopathies and Metabolic Disease
No full textInsulin resistance in skeletal muscle is the primary defect in type 2 diabetes (T2D). While current in vitro models utilize two-dimensional (2D) myotubes, these cultures express low levels of insulin-responsive GLUT4 and elevated levels of insulin-independent GLUT1, limiting their relevance for modeling insulin resistance. Human skeletal muscle microtissues, differentiated under uniaxial tension across two microposts, exhibit greater insulin responsiveness than 2D myotubes and are amenable to contractile stimulation. However, a microtissue-based model of T2D has not been developed. The motivation of this thesis was the establishment of a microtissue model to investigate T2D-associated impairments in force generation, myokine signaling, and protein synthesis, thereby establishing a multiplex platform for the study of dysregulation in T2D. First, to ensure accurate contractile assessments, we developed protocols to identify the electrical field stimulation parameters eliciting peak twitch and tetanus force from microtissues. The generalizability of these approaches was demonstrated across protocol users, micropost culture devices, and distinct myoblast cell line types. Our results revealed cell type-specific contractile properties, with all microtissues contracting and relaxing more slowly than native muscle. Next, to ensure accurate assessment of protein synthesis, we implemented stable isotope amino acid tracer to measure fraction-specific muscle protein synthesis rates in microtissues, demonstrating improved accuracy over puromycin-based methods. We uncovered that the myofibrillar protein fraction of microtissues is more stable than the sarcoplasmic fraction, a property of native human muscle absent in 2D myotubes. Finally, to evaluate the relevance of microtissues for modeling insulin resistance, we characterized their glucose transporter profile. Microtissues exhibited significantly higher glucose uptake potential than 2D myotubes, with a ~60-fold higher GLUT4:GLUT1 ratio. However, they required supraphysiological insulin concentrations to maintain functional integrity under conventional insulin-based differentiation protocols. Through this work, we demonstrated the need for an insulin-free differentiation media formulation to enable T2D modeling. In parallel, we benchmarked the functional and metabolic properties of microtissues relative to 2D myotubes and native muscle. These findings identify a hurdle for the development of a multiplex microtissue-based T2D model while also delivering a comparative framework to guide platform selection for the study of skeletal myopathies and metabolic disease.Ph.D
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