Investigation of environmental factors on the intranuclear landscape of mesenchymal stromal cells
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.
Includes bibliographical references.
Embargo Expires: 01/09/2025