Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
Browse
4 results
Search Results
Item THE IMPACT OF ENGINEERED MECHANICAL CONFINEMENT ON MESENCHYMAL STEM CELL AND LUNG FIBROBLAST MECHANOBIOLOGY(2020) Doolin, Mary; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mechanical cues have been shown to influence cell gene expression, cell protein expression, and cell behaviors critical for homeostasis and disease progression. Cells experience the mechanical cue of confinement in vivo, such as within the extracellular matrix, and in vitro, such as within tissue engineered scaffolds. Despite its prevalence, the impact of mechanical confinement on cell fate is poorly understood. Cues from the mechanical microenvironment are primarily sensed and responded to by the cytoskeleton, which transmits forces to the nucleus and can thereby alter gene expression. The nucleus itself is also a mechanosensor, sensing external forces and again altering gene expression. Mesenchymal stem cells (MSCs) and lung fibroblasts are known to be sensitive to mechanical forces, yet the effect of mechanical confinement on these cells is unclear. In this dissertation, we investigated how mechanical confinement induced by engineered microchannels influences MSC morphology and migration. Notably, we show that confinement alters the relative contributions of cytoskeletal and contractile machinery in MSC migration in unconfined and confined spaces. We next investigated how mechanical confinement induced by microchannels influences MSC and fibroblast nucleus volume. When certain cytoskeletal machinery was inhibited, nucleus volume was altered only in MSCs in wide channels, suggesting diverging roles of the cytoskeleton in regulating nuclear deformation and migration in different degrees of confinement and in different cell types. While performing this work, we observed a lack of assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. Therefore, we designed, created, and validated a confining micropillar assay that achieves these requirements. Using these confining micropillars, we investigated the effect of confinement on lung fibroblast to myofibroblast transition (FMT), a hallmark of idiopathic pulmonary fibrosis. Cell density was more predictive of FMT than the degree of confinement induced by micropillar arrays. These results improve our understanding of how MSCs and lung fibroblasts respond to confinement, which will aid in the rational design of MSC-based therapies and FMT-targeting therapies.Item VIMENTIN AND CYTOKERATIN INTERMEDIATE FILAMENTS IN THE MECHANOBIOLOGY AND MALIGNANT BEHAVIORS OF CHORDOMA CELLS(2018) Resutek, Lauren; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chordoma, an aggressive tumor derived from notochordal remnants, is difficult to treat due to its proximity to the spinal cord and brain stem and its resistance to conventional treatments, such as radiation and chemotherapy. The development of effective treatments requires research at the molecular level, which presumably due to its rare diagnosis, is lacking for chordoma. Recent studies have identified potential targets for systemic therapy; however, there are currently no drugs approved by the Food and Drug Administration (FDA) to treat chordoma. One promising approach is to target the cytoskeleton, in order to stall progression and sensitize cells to chemotherapeutics. Similar to other cancers, chordoma cells co-express vimentin and cytokeratin intermediate filaments (IFs), which have both been found to play roles in cell mechanical properties and behaviors and their expression has been associated with cancer metastasis, chemoresistance, and poor prognosis. Therefore, we investigated the functional roles of vimentin and cytokeratin IFs in chordoma cells using RNA interference (RNAi). First, we examined whether cytoskeletal disruption by siRNA-mediated silencing of vimentin or cytokeratin-8 altered the chordoma phenotype. We determined that the vacuolated cytoplasm, a distinguishing feature of chordoma, was dependent on cytokeratin-8 IFs. Next, we examined the effects of vimentin and cytokeratin-8 knockdown on chordoma cell mechanics. We found that chordoma cell stiffness, traction forces, and mechanosensitivity to substrate stiffness were all dependent on vimentin IFs. These results suggest that vimentin, rather than cytokeratin, IFs play a predominant role in chordoma cell mechanobiology. Finally, we analyzed the roles of vimentin and cytokeratin-8 IFs in cellular behaviors associated with cancer progression. We demonstrated that chordoma cell invasion and expression of the biomarker sonic hedgehog were dependent on vimentin. Further, we found that decreasing vimentin expression in chordoma cells may increase their sensitivity to chemotherapeutics. Because mechanical cues are important determinants of cell function, we hypothesize this correlation is in part due to the newly discovered role of vimentin IFs in chordoma cell mechanobiology. These results elucidate novel roles of vimentin and cytokeratin-8 IFs in chordoma cells, which may assist in the development of effective treatments for chordoma.Item The Influence of Vimentin Intermediate Filaments on Human Mesenchymal Stem Cell Response to Physical Stimuli(2017) Sharma, Poonam; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mesenchymal stem cells (MSCs) are increasingly being investigated as a therapeutic cell population for a variety of diseases. However, these therapies are limited by an imperfect understanding of how MSCs interact with and respond to their physical environment. Cell response to external stimuli is mediated by the cytoskeleton. Of the cytoskeletal proteins, understanding of vimentin intermediate filaments’ influence on MSC behavior is still lacking, despite increasing evidence that they are involved in many cellular processes. In this work, we investigated the influence of vimentin intermediate filaments in modulating MSC characteristics and behavior by using lentiviral shRNA transduction to decrease vimentin levels in MSCs through RNA interference. First, the contribution of vimentin intermediate filaments to the deformability of MSCs within agarose hydrogels was examined. Vimentin-deficient MSCs were found to be less deformable than control cell populations and this resistance to deformation may be due to the compensatory role of actin microfilaments. Next, to determine how vimentin affects the ability of MSCs to interact with various microenvironments, we examined cell spreading on different extracellular matrix proteins, multiple substrate stiffness’, and in response to fluid shear stress. An intact vimentin network was found to be necessary for unimpaired spreading on fibronectin, but only on stiffer substrates. Further, vimentin appears to be involved in resisting cell area changes in response to low fluid shear stress. Vimentin’s physical interaction with focal adhesions, rather than an impact at the transcriptional or translational level, may contribute to the cell spreading response observed. Finally, in the third part of this work, we examined the influence of vimentin on chondrogenic differentiation of MSC populations. Unexpectedly, we found that vimentin may not be involved in chondrogenic differentiation in late stage chondrogenic cultures. Instead, the culture condition-dependent microenvironment may have a greater impact, particularly in gene expression of matrix degrading enzymes and the αV integrin subunit. Altogether, these studies indicate a role for vimentin in the MSC response to physical stimuli. Moreover, this work furthers the dialogue surrounding MSCs’ interaction with different environments, the understanding of which will be critical for the development and evaluation of cell-based therapies.Item Cytoskeletal Mechanics and Mobility in the Axons of Sensory Neurons(2011) Chetta, Joshua; Shah, Sameer B; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The axon is a long specialized signaling projection of neurons, whose cytoskeleton is composed of networks of microtubules and actin filaments. The dynamic nature of these networks and the action of their associated motor and cross-linking proteins drives axonal growth. Understanding the mechanisms that control these processes is vitally important to neuroregenerative medicine and in this dissertation, evidence will be presented to support a model of interconnectivity between actin and microtubules in the axons of rat sensory neurons. First, the movement of GFP-actin was evaluated during unimpeded axonal outgrowth and a novel transport mechanism was discovered. Most other cargoes in the axon are actively moved by kinesin and dynein motor proteins along stationary microtubules, or are moved along actin filaments by myosin motor proteins. Actin, however, appears to be collected into short-lived bundles that are either actively carried as cargoes along other actin filaments, or are moved as passive cargoes on short mobile microtubules. Additionally, in response to an applied stretch, the axon does not behave as a uniform visco-elastic solid but rather exhibits local heterogeneity, both in the instantaneous response to stretch and in the remodeling which follows. After stretch, heterogeneity was observed in both the realized strain and long term reorganization along the length of the axon suggesting local variation in the distribution and connectivity of the cytoskeleton. This supports a model of stretch response in which sliding filaments dynamically break and reform connections within and between the actin and microtubule networks. Taken together, these two studies provide evidence for the mechanical and functional connectivity between actin and microtubules in the axonal cytoskeleton and suggest a far more important role for actin in the development of the peripheral nervous system. Moreover this provides a biological framework for the exploration of future regenerative therapies.