Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Developmental Biology

Supervisor

Hess, David A.

Affiliation

Robarts Research Institute

Abstract

Regenerative medicine is a multidisciplinary effort to regenerate or replace a deficiency of functional cells/tissues, such as deteriorated vasculature in cardiovascular pathologies or depleted -cell mass in diabetes. The transplantation of pro-regenerative progenitor cell populations has demonstrated therapeutic benefits in pre-clinical models, although translation to clinical efficacy is limited by the paradoxical balance between cell expansion ex vivo and loss of regenerative functions in vivo. Herein, this thesis encompasses two distinct approaches to circumvent translational deficiencies; 1) prevention of hematopoietic progenitor cell differentiation during ex vivo expansion and 2) utilizing the secretome of pancreas-derived multipotent stromal cell as a biotherapeutic agent. Across the first two studies, I investigated whether the inhibition of retinoic acid (RA)-signaling would limit HPC differentiation and retain pro-vascular and islet regenerative functions in vivo. These studies identified a bias of HPC towards megakaryopoiesis with prolonged culture; however, also demonstrated that the reversible inhibition of RA-signaling enhances the expansion of HPC progeny that retain a primitive phenotype and multifaceted pro-regenerative functions in vivo. Albeit, cell transplantation is limited by transplanted cell survival and subsequent secretion of pro-regenerative stimuli. Accordingly, we have demonstrated the transplantation of conditioned media generated by therapeutic cells by-passes these limitations while stimulating endogenous mechanisms of tissue regeneration. To this end, I isolated a heterogenous Panc-MSC population during culture of human islets in vitro. Using label-free mass spectrometry and flow cytometry analyses, I provide the first in-depth characterization of human Panc-MSC in comparison to BM-MSC. Specifically, I demonstrated the proteome of Panc-MSC restricts adipogenesis and RA-signaling; albeit, is competent to pro-neural stimuli and primed for robust expansion ex vivo. Next, I determined that Panc-MSC secrete pro-angiogenic and islet regenerative stimuli which are harbored within extracellular vesicles. Collectively, this body of work provides novel insights towards multifaceted therapeutic cell populations and lays a foundation to explore the cell-free biotherapeutics for applications of regenerative medicine.

Summary for Lay Audience

This body of work undertakes a broad experimental approach to identify “stem cell” populations cells from human post-natal or adult tissues, such as umbilical cord blood, bone marrow, or pancreas. Specifically, I sought to identify cells which may be used as a biological agent to activate endogenous (self-) regeneration to treat diabetes and life-threating complications of blood vessels. In diabetes, a loss or dysfunction of insulin-producing β-cells leads to elevated blood sugars that damage blood vessels in organs throughout the body. In the first half of this thesis, I developed a method to increase the number of blood lineage-specific stem cell populations that can regenerate beta-cells and blood vessels in mouse models. Albeit, I present early evidence that cells of the megakaryocyte (platelet-producing)-lineage may have therapeutic potential. The second half of this thesis characterized a mesenchymal progenitor population from human pancreas tissue using high-sensitive equipment to detect over >7000 proteins in an effort to establish a “proteomic fingerprint” of therapeutic cell populations. Accordingly, I proceeded to show that cell transplantation can be avoided, and media conditioned by mesenchymal progenitors can be concentrated for use as a biological cocktail to stimulate blood vessel and β-cell regeneration in mouse models. This body of work provides foundational evidence to further explore the use of post-natal and adult stem/progenitor cell populations as a therapeutic tool to treat diabetes and/or vascular complications.

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