Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Hess, David A.

Abstract

Cellular transplantation strategies have aimed to combat critical limb ischemia (CLI) by inducing endogenous blood vessel regeneration. Transplantation of mesenchymal stromal cells (MSC) for CLI has proven safe and well-tolerated but demonstrated only modestly improved clinical outcomes explained in part by a lack of MSC survival and/or potency in ischemic tissues. This thesis focuses on the development of improved transplantation strategies for patients with CLI utilizing purified pro-angiogenic subsets of MSC and endothelial colony forming cells (ECFC) seeded within decellularized adipose tissue (DAT) bioscaffolds. First, I investigated whether purification of bone marrow-derived MSC based on a conserved stem cell function, high ALDH-activity, could isolate a pro-vascular MSC subset after culture. Conditioned media generated by ALDHhi MSC promoted endothelial cell proliferation, survival, and tube forming functions under serum-free conditions, and a robust recruitment of mouse derived endothelial cells was observed after subcutaneous implantation of ALDHhi MSC within immunodeficient mice. Interestingly, isolating ECFC based on high ALDH-activity did not improve endothelial cell functions. Instead, isolation of ECFC expressing the progenitor cell surface marker, CD34 purified for ECFC with a mature endothelial cell surface phenotype and diminished colony- and tubule-forming functions in vitro. Before transplanting ECFC and MSC within human DAT scaffolds, I sought to develop robust methodologies to extract cells from DAT scaffolds, without generation of autofluorescent debris that impaired flow cytometric cellular evaluations. After protease dissociation, DAT scaffolds were centrifuged using a density gradient to produce samples with >99% purity. This method enabled accurate quantification of changes in ECFC surface marker expression alongside a decline in ECFC proliferation when co-cultured with MSC on DAT scaffolds in vitro. After intramuscular injection, or DAT scaffold implantation of ECFC and/or MSC into NOD.SCID mice with acute unilateral hindlimb ischemia, the level of blood perfusion in the ischemic limb was unaffected compared to saline injection. Importantly, implanted DAT scaffolds became vascularized and enhanced survival of ECFC or MSC. Taken together, this body of work indicates the importance of purifying pro-regenerative subsets to maximize cellular potency for clinical benefit.

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