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Browsing by Author "Ghosh, Soham, advisor"

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    Characterization of chromatin remodeling in mesenchymal stem cells on the application of oxidative stress
    (Colorado State University. Libraries, 2022) Kabi, Neda, author; Ghosh, Soham, advisor; Popat, Ketul, committee member; Goodrich, Laurie, committee member; Johnstone, Brian, committee member
    Chromatin is a highly dynamic entity of the eukaryotic cell nucleus. Contrary to previous belief that chromatin maintains a well-defined permanent architecture in the interphase nucleus, new evidences are emerging with a support of the notion that chromatin can locally and globally rearrange itself to adapt with the cellular microenvironmental changes. Such microenvironmental changes can be related to biophysical such as change in the stiffness of extracellular matrix or the force applied on the cell as well as biochemical such as change in the oxidative stress, osmolarity or the pH. It is not well understood how the chromatin architecture changes under such environmental changes and what is the functional significance of such change. Characterization and quantification of chromatin remodeling is therefore a first step to understand the chromatin dynamics for elucidating complex subnuclear behavior under the influence of single or multiple environmental changes. Towards that end, in this work, human bone marrow derived mesenchymal stem cells were used to characterize such chromatin level changes under the changing oxidative stress on the cells. Oxidative stress was applied using hydrogen peroxide treatment. After validation of the application of oxidative stress, a series of experiments and subsequent analysis was performed to understand the hallmarks of chromatin remodeling at high spatiotemporal resolution. Specific chromatin remodeling pattern was observed in the heterochromatin, euchromatin and the interchromatin regions. Finally, a key component of chromatin remodeling complex called ARID1A was identified which is critical for the chromatin remodeling process.
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    Computational methods for the analysis of cell migration and motility
    (Colorado State University. Libraries, 2024) Havenhill, Eric Colton, author; Ghosh, Soham, advisor; Heyliger, Paul, committee member; McGilvray, Kirk, committee member; Zhao, Jianguo, committee member
    Collective cell migration (CCM) is necessary for many biological processes, such as in the formation or regeneration of tissue, fibroblast movement in wound healing, and the movement of macrophages and neutrophils in the body's immune response, to name a few. CCM is commonly modeled with PDEs, however these equations usually model the population density, rather than the displacement field describing the movement of any arbitrary cell. One unknown aspect of this movement is the various methods that cells use to facilitate communication to each other. Chemical communication plays a substantial role in directed cell movement, however, other mechanical methods, such as the propagation of stresses through a shared substrate to neighboring cells and cell behavior in a crowded environment, also play an important role which is less understood. The quantification of the kinematic and dynamic characteristics in CCM would present several novel advancements in understanding the collective cell behavior. First, the dynamic mode decomposition (DMD) framework is utilized. DMD allows for the recovery of a dynamic system, in the form of an ODE or PDE, by sampling the states of a system. In the context of the cell migration, the displacements of fibroblasts during a scratch-wound assay are obtained, which result in a governing PDE through the DMD process. This PDE is used in conjunction with modern optimal control theory to develop a 2D and 3D trajectory for the migration of controllable cells to a target. On an individual level, with the hybrid use of modern static structural optimization and simple non-linear control, a cell's cytoskeleton during migration can be studied, providing for the quantification of the traction force exerted on the substrate. The results of this analysis are compared with stress and structural optimization models in ANSYS and FEBio, which uses the finite element method, so that a reasonable range of these stresses during CCM can be provided. To further study the individual mechanics of cell migration, the proposed hybrid model is extended to a fully dynamic model which predicts the cytoskeletal stress fiber formations over time that require the minimal amount of material with the use of optimal control theory. The results of this research could provide useful applications in many real-world situations, from the generating of a trajectory for microrobots during drug delivery to the study of the collective migration of organisms including cells.
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    Investigation of environmental factors on the intranuclear landscape of mesenchymal stromal cells
    (Colorado State University. Libraries, 2022) Kaonis, Samantha, author; Ghosh, Soham, advisor; Johnstone, Brian, committee member; Popat, Ketul, committee member; Dow, Steven, committee member
    Mesenchymal stromal cells (MSC), also known as mesenchymal stem cells, are popular candidates for tissue engineering and regenerative medicine. They can differentiate into many tissue types, and they can also help in regeneration through their trophic and immunomodulatory properties. Despite being investigated thoroughly for the last four decades and being under clinical trial in more than a thousand FDA approved studies, their application in clinics is very limited. One of the most important challenges in using MSC is that after harvesting from the patient, they need to be expanded to millions of cells for successful clinical outcomes. During this process, MSCs lose their differentiation potential, and trophic and immunomodulatory properties. In this thesis, I investigated the potential mechanisms of how environmental factors cause the MSC to divert from their phenotype during the expansion process. Subsequently, I intervened these mechanisms to achieve high quality MSCs without compromising the number of cells, i.e., their proliferation potential. Specifically, I investigated how two critical biophysical factors - mechanical stiffness and oxygen concentration of the MSC environment affects the cell phenotype and function through mechanisms involving epigenetic modifiers, transcription factors, and the chromatin architecture. First, the regulation of mechanics-induced population heterogeneity in MSCs was examined. Plastic culture and fibrotic conditions post-transplantation experienced by the MSC is completely different from the natural biomechanical niche of the MSC. Accordingly, the role of the mechanical environment has been shown to be a critical determinant of MSC gene expression and function. In this study, we report that human bone marrow-derived primary MSC population becomes phenotypically heterogenous when they experience an abnormal mechanical environment, compared to their native environment. Using a newly developed technique to quantify the heterogeneity, we provide evidence of phenotypical heterogeneity of MSC through high-resolution imaging and image analysis. Additionally, we provide mechanistic insight into the origin of such substrate mechanics-driven heterogeneity, which is further determined by the cell-cell mechanical communication through the substrate. In the second study, we investigated how the chromatin architecture and epigenetic landscape changes in MSCs by the substrate mechanical stiffness, thus causing a shift from the MSC phenotype. Using high-resolution confocal microscopy and advanced image analysis we identified the key epigenetic drivers in the mechanical stiffness mediated chromatin organization changes. Subsequently, we targeted several components of a proposed mechanobiological pathway to achieve MSCs with higher growth factor secretion without compromising their proliferation. The outcome of these studies might provide mechanism-driven design principles to the molecular, cellular and tissue engineering researchers for the rational design of MSC culture conditions and scaffolds, thus improving their functional outcome. Finally, the effect of oxygen concentration on MSC proliferation and performance wereexplored. Culture under physiological oxygen concentration (physioxia) can increase the proliferation of MSCs through a pathway initiated by the stabilization of the hypoxia-inducible factor-1 (HIF-1). Stabilized HIF-1α translocates into the nucleus, triggering the transcription of target genes conducive to MSC activity and proliferation. However, stabilized HIF-1α also triggers the p21 pathway causing cell cycle arrest, decreasing the MSC proliferation thereby limiting the beneficial effect of physioxia. Maintaining low oxygen conditions can be challenging, especially at a large scale, so rational exploitation and selective manipulation of such pathways through biochemical means has the potential to culture MSCs easily at scale. In this work, we created a mathematical model to predict optimal physioxic culture parameters to achieve the highest MSC proliferation. Through analysis of a gene downstream of the HIF-1 pathway, we also compared standard physioxic culture (2% O2) to treatment with deferoxamine mesylate (DFO), a physioxia-mimicking drug. The outcomes of this study might provide the rationale for MSC culture under standard hyperoxic conditions with only a simple addition of a combination of drugs to the culture medium to improve the scalability of MSC culture. Together, the results of the work will identify the mechanistic details of culture environment factors that play a role in determining the phenotype of MSCs during in vitro expansion process. The combination of these techniques to optimize MSC culture in vitro has the potential to resolve the current impediment to the clinical success of MSC therapies.