Browsing by Author "Johnstone, Brian, committee member"

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Open Access
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.
Embargo
Developing preclinical models for intervertebral disc degeneration: analyzing mechanical, molecular, and immunological interventions
(Colorado State University. Libraries, 2024) Bonilla, Andres, author; Easley, Jeremiah, advisor; Dow, Steve, committee member; Puttlitz, Christian, committee member; Johnstone, Brian, committee member
Low back pain is a prevalent global health issue, significantly affecting quality of life and contributing to economic burdens through reduced productivity and healthcare costs. Intervertebral disc degeneration (IVDD) is recognized as a major underlying cause of chronic low back pain. Despite advances in therapeutic strategies for IVDD, the translation of these treatments from preclinical research to clinical application remains challenging due to the lack of appropriate animal models that accurately mimic the complex pathophysiology of human IVDD. This document aims to address these limitations by developing and validating novel ovine and immune-induced animal models of IVDD, and to provide insights into the molecular, biomechanical, and immunological aspects of the disease. Chapter 1 introduces the clinical significance of low back pain and the central role of IVDD in its development. The chapter highlights the unmet need for robust preclinical models to facilitate the evaluation of potential therapeutic interventions. Chapter 2 reviews the existing literature on ovine models of IVDD, emphasizing their relevance to human spinal disorders due to the anatomical, physiological, and histological similarities between sheep and humans. Ovine models are particularly valuable for studying both spontaneous and induced IVDD, providing a critical platform for translational research. Chapter 3 presents a groundbreaking study that conducts the first comparative analysis of surgical, imaging, histological, and proteomic characteristics between cervical and lumbar intervertebral discs (IVDs) in an ovine model of IVDD. The results demonstrate a comparable progression of IVDD in both regions, challenging the longstanding emphasis on lumbar IVDs in research and underscoring the importance of cervical models in advancing our understanding of the disease. These findings have substantial clinical and research implications, indicating that treatments traditionally developed and evaluated for lumbar IVDD may also be relevant for cervical pathology. Furthermore, the identification of specific biomarkers, could significantly enhance early diagnosis and inform the development of tailored therapeutic interventions. Chapter 4 introduces a novel model utilizing extracorporeal shock wave therapy (ESWT) applied to ovine IVDs. While no significant evidence of IVDD was observed during the 12-week study period, localized bone formation at the treatment sites was identified. This finding provides important insight into the effects of ESWT, suggesting that while it is conventionally used as a therapeutic modality, it may also have unintended consequences, such as promoting bone formation, which could potentially lead to tissue damage. These results highlight the need for further refinement of shock parameters to reliably induce progressive IVDD, offering valuable data for future research into both the therapeutic and adverse effects of ESWT in spinal treatments. In Chapter 5, a mechanical compression model utilizing MRI-compatible materials was developed to induce disc degeneration in ovine lumbar discs, marking the first report of its kind to our knowledge. This innovation allows for the longitudinal tracking of degeneration with a measurable rate of compression, leveraging MRI as the most critical tool for diagnosing IVDD. While the model successfully induced biomechanical changes, including reduced disc height and altered neutral zone dynamics, no significant histological or biochemical degeneration was observed. However, these findings provide valuable insights for future researchers using mechanically induced models, offering a foundation to refine and optimize the model for tracking the progression of degeneration more accurately. Chapter 6 explores the role of immune system in IVDD by developing an immune-induced model using a nucleus pulposus (NP) antigen vaccine. Rabbits were vaccinated against NP following IVD injury, leading to accelerated degeneration in comparison to non-vaccinated animals. This study highlights the potential of immune responses in accelerating disc degeneration, offering a novel avenue for understanding the interplay between immunity and IVDD progression. This document contributes to the advancement of IVDD research by establishing and validating novel animal models, including ovine, mechanical compression, and immune-induced models. The proteomic findings and biomechanical evaluations presented in this document offer critical insights into the molecular pathways involved in IVDD and lay the foundation for the development of customized therapeutic strategies. Future research should focus on refining these models to better replicate the complexities of human IVDD and explore long-term therapeutic interventions that can mitigate degeneration and restore disc function.
Open Access
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.