Browsing by Subject "Stem cell"
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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 Biomimetic hydroxyapatite/collagen composite drives bone niche recapitulation in a rabbit orthotopic model.(Materials today. Bio, 2019-03) Minardi, S; Taraballi, F; Cabrera, FJ; Van Eps, J; Wang, X; Gazze, SA; Fernandez-Mourev, Joseph S; Tampieri, A; Francis, L; Weiner, BK; Tasciotti, ESynthetic osteoinductive materials that mimic the human osteogenic niche have emerged as ideal candidates to address this area of unmet clinical need. In this study, we evaluated the osteoinductive potential in a rabbit orthotopic model of a magnesium-doped hydroxyapatite/type I collagen (MHA/Coll) composite. The composite was fabricated to exhibit a highly fibrous structure of carbonated MHA with 70% (±2.1) porosity and a Ca/P ratio of 1.5 (±0.03) as well as a diverse range of elasticity separated to two distinct stiffness peaks of low (2.35 ± 1.16 MPa) and higher (9.52 ± 2.10 MPa) Young's Modulus. Data suggested that these specific compositional and nanomechanical material properties induced the deposition of de novo mineral phase, while modulating the expression of early and late osteogenic marker genes, in a 3D in vitro model using human bone marrow-derived mesenchymal stem cells (hBM-MSCs). When tested in the rabbit orthotopic model, MHA/Col1 scaffold induction of new trabecular bone mass was observed by DynaCT scan, only 2 weeks after implantation. Bone histomorphometry at 6 weeks revealed a significant amount of de novo bone matrix formation. qPCR demonstrated MHA/Coll scaffold full cellularization in vivo and the expression of both osteogenesis-associated genes (Spp1, Sparc, Col1a1, Runx2, Dlx5) as well as hematopoietic (Vcam1, Cd38, Sele, Kdr) and bone marrow stromal cell marker genes (Vim, Itgb1, Alcam). Altogether, these data provide evidence of the solid osteoinductive potential of MHA/Coll and its suitability for multiple approaches of bone regeneration.Item Embargo Cellular Ensembles in Alveolar Homeostasis and Repair(2023) Konkimalla, ArvindLung epithelium, the lining that covers the inner surface of the respiratory tract, is directly exposed to the environment and thus susceptible to airborne toxins, irritants, and pathogen-induced damages. In adult mammalian lungs, epithelial cells are generally quiescent but can respond rapidly to repair damaged tissues. Evidence from experimental injury models in rodents and human clinical samples has led to the identification of these regenerative cells, as well as pathological metaplastic states specifically associated with different forms of damages. The primary alveolar stem cell, alveolar type-2 cells (AT2s) are sparsely distributed and make up only 5% of the surface area. Despite this organization, AT2s are still able to maintain tissue homeostasis and achieve efficient repair after injury. However, the underlying mechanisms of stem cell activation, injury response, and subsequent cell-cell communication signals mediating resolution of injury and restoration of alveolar homeostasis remain elusive. Additionally, modulation of these regenerative cells for therapeutic potential has not been well established, primarily due to a lack of viable gene editing tools and vehicles for gene delivery to the alveolar stem cells, and neighboring niche. First, to better target the lung alveolus, we screened and identified cell-type specific adeno-associated virus (AAV) serotypes, enabling efficient targeting and gene expression of exogenous genes in alveolar stem cells as well neighboring mesenchyme. These tools were also capable for both in vitro and in vivo gene editing, forgoing the need for development of complex genetic mouse models as well as enabling diverse, viral-based screens. Second, using 3-dimensional, thick tissue imaging we reveal that a single AT2 cell can enface multiple alveolar compartments by virtue of a unique, multi-apical domain architecture. Lineage tracing and live imaging coupled with genetic and AAV-mediated selective ablation of AT2s was used to show robust recovery of AT2 numbers and distribution via clonal proliferation and migration, even after three successive rounds of ablation. Clonal tracing revealed that a single AT2 can differentiate to cover multiple alveolar cups. During injury repair, AT2s dynamically reorganize their apical domains to facilitate either migration or differentiation. Single- cell transcriptome profiling, genetic and pharmacologic disruption of actin dynamics, and evaluation of multiple physiologically relevant disease states identified the roles of actin cytoskeleton, cell migration, and multi-apical domains in AT2 recovery and regenerative potency. Lastly, using cell-type specific ablation of alveolar type 1 cells (AT1s), we identified novel mechanisms of epithelium-mediated signaling to mesenchymal fibroblasts, thereby uncovering novel mechanisms of fibrosis initiation and progression. Modulation of AT1 ablation dynamics preferentially drives fibrosis or, in contrast, emphysema, both at histological and physiological levels, as assessed by whole body plethysmography. Single-cell sequencing identified the epithelial and mesenchymal cell identities involved in regenerative processes, as well as identification of a PDGFA signaling axis between AT1s and resident alveolar fibroblasts necessary for fibroblast maintenance. We demonstrate that modulation of these signaling pathways during lung regeneration could enhance fibrosis or convert fibrosis to emphysema. In sum, the work presented herein both develops functional tools for perturbation of alveolar stem cells, as well as an improved understanding of alveolar architecture, stem cell dynamics during injury repair, homeostatic intercellular signaling, and mechanisms of disease progression.
Item Open Access Stem Cell-Based Strategies to Study, Prevent, and Treat Cartilage Injury and Osteoarthritis(2012) Diekman, Brian O'CallaghanArticular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.
Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds.
We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFRα+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA.
Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect.
The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.