Date of Award

12-1-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Environmental Health Sciences

First Advisor

Michael Laiosa

Committee Members

Kurt Svoboda, Ava Udvadia, Hongbo Ma, Jeanette Vasquez-Vivar

Abstract

The cells of the immune system are descended from multipotent hematopoietic stem cells (HSCs) that emerge during development. Multipotency means that a single progenitor HSC can differentiate into any cell of the immune system. HSCs are required to do this for the lifetime of the organism through a process called self-renewal, and as such, any perturbation during development or in the bone marrow can have a trickle-down effect, affecting the self-renewal capacity or ability to terminally differentiate. The aryl hydrocarbon receptor (AHR) is a known regulator of HSCs. The AHR is a transcription factor required for the detoxification of numerous xenobiotics and is agonized by the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p¬-dioxin (TCDD). The advent of AHR-/- animal models have furthermore demonstrated that the AHR has an important role in the regulation of HSCs.

The overall goal was to characterize the effect of AHR agonism on long-term HSCs (LT-HSCs) – the cells at the top of the HSC hierarchy required for long-term, lifelong reconstitution. Our approach was to utilize a lethal irradiation reconstitution model to compete vehicle treated HSCs against developmentally exposed TCDD HSCs.

During the course of this investigation, we sought further characterization of the effects of developmental AHR activation and absence on HSCs. Our approach was to compare AHR agonism and absence in HSCs across anatomical locations and developmental time points, and probe the differences in reactive oxygen species production and mitochondrial mass.

Finally, given the inadequacies of using surface markers alone, we sought to further elucidate how flow cytometry could be used to gather a population of HSCs that is more enriched for LT-HSCs. For this, we reframed how LT-HSCs are identified using flow cytometry and leveraged existing knowledge about their physical characteristics.

Taken together, the data presented herein supports 3 conclusions; 1: Developmental AHR agonism with TCDD decreases the repopulating ability of LT-HSCs; 2: AHR agonism and absence exhibit different effects on HSCs depending on developmental time and anatomical location, and 3: sorting HSCs based on surface markers in combination with selecting for low mitochondrial membrane potential, low mitochondrial mass, and low reactive oxygen species production can enrich for LT-HSCs better than using surface markers alone.

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