Aromatics & Exosomes: The Translation of Cell Therapy Restricted; Files Only

Lewis, Holly (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/gt54kn754?locale=en
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Abstract

Abstract

Aromatics & Exosomes: The Translation of Cell Therapy

By

Holly C. Lewis

Mesenchymal stromal cells (MSCs) are a low-frequency population in the adult bone marrow. These self-renewing pluripotent stem cells can be easily expanded ex vivo, generating clinical quantities of personalized cell therapeutics. Despite showing biologic efficacy in a variety of mammalian studies and clinical trials, the mechanisms by which MSCs exert their bioactivity have been incompletely described.

One of the principle mechanisms we and others have shown as crucial for MSC efficacy is the enzyme indoleamine 2,3-dioxygenase (IDO). This enzyme catalyzes the key reaction in tryptophan metabolism. The synthetic drug 1-methyl tryptophan is a selective inhibitor of IDO enzymatic activity that is being tested in cancer immunotherapy trials, particularly for patients with IDO+ tumors. Based on its chemical structure, we hypothesized 1MT might also activate the aryl hydrocarbon receptor (AHR). AHR is a widely-expressed transcription factor that is classically understood as the receptor for 2,3,7,8- tetrachlorodioxin, a potent environmental toxin. Such a mechanism of action for 1MT suggests its application for a wider range of patients, irrespective of tumor IDO expression. Such observations support a novel paradigm by which AHR-activating compounds like 1MT may be used in cancer immunotherapy to stimulate a pro-inflammatory response.

Collaborations with our lab have recently shown that MSC-conditioned culture medium (CM) can maintain healthy peripheral-blood-derived antibody-secreting cells (ASCs) for up to 30 days in vitro. We hypothesized that some of this in vitro support was due to nanoscale extracellular membrane vesicles, or exosomes. We interrogated exosome production from replicating and irradiated, growth- arrested MSCs to model the physiology of endogenous-mobilized or quiescent marrow MSCs. We found that exosomes were able to reproduce the in vitro support to ASCs observed with unfractionated CM. Purified exosomes from both replicating and growth-arrested MSCs were comparable in their ability to support ASCs. To elucidate factors accounting for the in vitro ASC support, we performed proteomics on exosomes derived from replication-competent and growth- arrested MSCs, identifying factors involved in the vesicle-mediated delivery of immune signaling proteins. Taken together, these findings indicate that MSC-derived exosomes can serve as a model for cell-free cell therapy.

Table of Contents

Table of Contents

Foreword: Page 1

Chapter 1: An Introduction to Mesenchymal Cell Therapy Pages 5-42

Chapter 2: AHR Signaling as a Model for MSC Bioactivity Pages 44-63

Chapter 3: Modeling AHR Ligation In silico Pages 84-86

Chapter 4: Community Approaches for Diverse Cell Donorship Pages 88-92

Chapter 5: Modeling MSC-Based Therapies with Exosomes Page 95-115

Chapter 6: Conclusions and Next Steps Pages 134-146

References Pages 149-187

List of Figures

Chapter 1 Page 43

Figure 1: Licensing of MSCs Activates their Immunomodulatory Capabilities

Chapter 2 Pages 64-83

Figure 1: IDO and AHR expression in resting and IFN-γ-stimulated MSC treated with 1MT

Figure 2: AHR nucleotransloaction in MSCs treated with 1MT and AHR agonists

Figure 3: Known AHR ligands and Trp derivatives activate the AHR response in MSCs

Figure 4: Interferon-γ licensing of MSCs and AHR response

Figure 5 : RNA-seq analysis of 1MT and TCDD treated MSCs

Figure 6 Supplementary Table 1

Figure 7 Supplementary Table 2

Figure 6 Supplementary Table 3

Chapter 3 Page 87

Figure 1: In Silico Modeling of AHR Ligation

Chapter 4 Pages 93-94

Figure 1: Translational Stem Cell Therapy

Figure 2: Sickle & Flow Event June 18, 2016.

Chapter 5 Pages 116-133

Figure 1: MSC CM maintains in vitro ASC survival, but is abrogated by lipid-disruption

Figure 2. Electron microscopy shows CM from irradiated MSCs contains a greater number of exosome-sized extracellular vesicles

Figure 3. Immuno-gold electron microscopy confirms presence of known MSC-derived exosome markers

Figure 4. Highly-specific ELISA shows that Irradiated and Non-irradiated MSCs release the same quantity of CD9-positive exosomes

Figure 5: Exosomes from MSCs support ASC function irregardless of irradiation status

Figure 6: Exosome proteomics reveals high significance for exosome-mediated delivery of integrin signaling proteins

Figure 7: Supplemental FlowChart for Generation of Exosomes from MSCs

Figure 7 Supplementary Table 1

Figure 8 Supplementary Table 2

Chapter 6 Pages 147-148

Figure 1: Exosomes from Rhesus Macaques

Figure 2: Exosomes from AHR+ MSCs

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