Date of Award
8-1-2023
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Environmental Health Sciences
First Advisor
Michael D Laiosa
Committee Members
Kurt Svoboda, Janis Eells, Todd Miller, Dean Nardelli
Keywords
aryl hydrocarbon receptor, ex vivo culturing, hematopoietic stem cell
Abstract
Hematopoiesis, the process of blood and immune cell generation, is dependent upon a rare population of cells, hematopoietic stem cells (HSC) that reside in the bone marrow. HSCs have two critical functions, self-renewal and multi-lineage differentiation. The maintenance of normal hematopoiesis conditions, and ultimately normal immune system function, is dependent upon HSCs. Therefore, self-renewal is extensively regulated by the complex bone marrow microenvironment and its interactions with a wide array of intrinsic and extrinsic factors. The importance of functional HSCs on immune system function has resulted in the development of a growing spectrum of ex vivo systems to model hematopoiesis optimized for an array of biotechnological applications including toxicological assessments, clinical research, and pharmaceutical development. However, current ex vivo systems have extensive limitations in modeling self-renewal.Existing approaches have been optimized for primary bone marrow HSCs grown with defined growth factors and/or accessory cells in a traditional incubator. However, it is well known that self-renewal is dependent upon low O2 tension, and as such most current ex vivo systems remain insufficient in their ability to maintain HSCs due to the inappropriate growth conditions as cultures are maintained in ambient air. These practices have resulted in a persistent barrier in progressing the field forward. Thus, the overall objective was to develop an ex vivo system that enhances the maintenance of HSCs under physiologically relevant conditions that could be used for more reliable toxicological assessments. We hypothesized that high O2 tension is a significant confounder in toxicological studies as it diminishes HSC maintenance ex vivo and may modulate sensitivity to environmental exposures. We systematically developed an ex vivo system that allows for normal environmental regulation of hematopoiesis. This was done by adapting an existing expansion protocol in physioxic conditions. With our developed system, we were able to increase HSC enrichment and maintenance in physiologically relevant conditions. To assess the role of O2 tension on toxicant sensitivity, we exposed cultures with low doses of 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD), a well characterized immunotoxicant that mediates toxicity via the basic helix-loop-helix transcription factor referred as the aryl hydrocarbon receptor (AhR). We found that cultures maintained in physioxia (5% O2) had enhanced sensitivity to TCDD compared to cultures maintained in ambient air. This data implies that physioxic conditions may reveal profound differences in biological responses to toxicants compared to room air. Moreover, these findings have larger implications in toxicology as data generated from ex vivo studies are a critical component in toxicological risk assessments that are used to set guidelines for safe exposure levels. Moving forward, new guidelines should be implemented to ensure physioxic conditions are the standard for ex vivo studies, as this data ultimately impacts human and environmental health.
Recommended Citation
Rogan, Shana, "Accurate Risk Assessments Based on Blood and Immune Cell Progenitor Responses to Toxicological Exposures Requires Physiological Oxygen Levels in the Culture Microenvironment" (2023). Theses and Dissertations. 3334.
https://dc.uwm.edu/etd/3334