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<p> Downregulation of E-cadherin is a frequent event in epithelial cancers and it correlates with weakened cell-cell adhesion and the induction of an epithelial-to-mesenchymal transition (EMT). It is postulated that E-cadherin downregulation liberates the catenin p120 and allows p120's translocation to the nucleus where it interacts with and functionally regulates the novel BTB/POZ transcription factor, Kaiso. Kaiso mediates transcriptional repression of various tumourigenesis-associated genes via methylated CpG dinucleotides or a sequence-specific Kaiso binding site (KBS). The Kaiso/p120 interaction has been detected in E-cadherin expressing cells of various origins, but is seldom detected in N-cadherin expressing cells or cells that have undergone EMT. We hypothesize that p120 and Kaiso play a role in EMT by modulating the expression of EMT-associated genes. We demonstrated that TGF-β-induced EMT occurs in a dose- and time-dependent manner in NMuMG cells but not in FHL-124 cells. In both cells lines, the Kaiso/p120 interaction occurred irrelevant of EMT induction by TGF-β. In NMuMG cells, the expression of p120 increased with EMT induction, while the expression of Kaiso remained unchanged. Finally, misexpression of Kaiso and p120 in mammary epithelial cells affected TGF-β-mediated EMT induction by delaying the upregulation of the positive mesenchymal markers, N-cadherin and α-SMA.</p> / Thesis / Master of Science (MSc)

Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses. / Doctor of Philosophy / Keratins and keratin hydrogels may exhibit several properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for payloads for tissue engineering purposes. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles that were analyzed based on drug loading characteristics. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs). Finally, we tested these products to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Particles with a porous structure were obtained. The microstructure of these particles may lend them to applications in drug release or other payload delivery. Data showed an ability to load and unload specific drug payloads. Dense cell populations were loaded onto particles. Finally, studies show that both keratin and BM-MSCs support skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.

Eukaryotic cell chemotaxis, or directed cell migration in response to a chemoeffector gradient, plays a central role in many important biological process such as wound healing, cancer metastasis, and embryogenesis. In vivo, cells migrate on fibrous ECM, but chemotaxis studies are typically conducted on flat substrates which fail to recapitulate ECM or 3D gel environments with heterogeneous and poorly defined biophysical properties. To address these challenges, this thesis focused on developing a microfluidic assay device which utilizes a reductionist approach to study single cell chemotaxis on aligned, suspended ECM-mimicking nanofibers. The device is comprised of a network of microfluidic mixing channels which produce a temporally invariant, linear chemical gradient over nanofiber scaffolds in an observation channel. The microfluidic device design was guided by a numerical model and validated with experimental testing. This device was used to study mouse embryonic fibroblast NIH/3T3 response to platelet derived growth factor (PDGF) on flat polystyrene and suspended, polystyrene nanofibers with small (15 μm), and large (25 μm) spacing. Cell aspect ratio is lowest for flat polystyrene (spread morphology) and highest for large-spaced fibers (spindle morphology). Cells migrating on fibers begin to show a chemotaxis response to a PDGF gradient 10 times shallower than that required for chemotaxis response on a flat substrate. Furthermore, cells with spindle morphology maintain a robust and strong response over a broad range of chemoattractant concentration. These cells also had a 45% increase in speed and 26% increase in persistence over cells on flat polystyrene. The findings of this thesis suggest that 2D substrates may not be sufficient for studying physiologically relevant chemotaxis. / Master of Science

Tendon injuries are a significant cause of morbidity in performance horses with superficial digital flexor tendon injury reported to represent up to 43% of overall Thoroughbred racehorse injuries. Natural repair is slow and results in inferior structural organization and biomechanical properties and, therefore, reinjury is common. The inability of tendon to regenerate after injury, or to heal with mechanical properties comparable to the original tissue, is likely attributable to low vascularity and cellularity of the tissue, low number of resident progenitor cells, and healing under weight-bearing conditions. Strategies to improve tendon healing have focused on enhancing the metabolic response of tenocytes, modulating the organization of the newly synthesized extracellular matrix, or administering progenitor cells to enhance repair. Significant research effort has been directed at the use of adult mesenchymal stem cells as a source of progenitor cells for equine tendon repair and recent clinical applications have utilized adult autologous stem cells derived either from adipose tissue or bone marrow aspirates. Isolation of a homogenous population of stem cells from bone marrow is time-consuming, and there is much variation in cell numbers, cell viability and growth rates among samples. Recently, a population of progenitor cells has been isolated from equine flexor tendons, thus providing an alternative source of progenitor cells from the target tissue for therapeutic intervention. The interaction between cells and the extracellular matrix (ECM) is an important factor in regulation of cell function. Proliferation, migration, differentiation and gene expression of many cell types are altered by adhesion to and interaction with matrix proteins and the extracellular environment. Tendon progenitor cells reside within a niche that comprises primarily parallel collagen fibers, and this niche plays an important role in regulating their function and differentiation. Culture conditions replicating this environment could be beneficial for both cell growth and matrix gene expression. The objectives of the study were to compare cell growth kinetics and biosynthetic capabilities of bone marrow mesenchymal stem cells (BMMSCs) and tendon derived progenitor cells (TPCs) cultured on commercially available bovine, highly purified bovine, porcine, and rattus collagen sources and standard tissue culture surfaces. We hypothesized that collagen type I matrix would preferentially support TPC proliferation and up regulate gene expression for collagens and organizational components of tendon and therefore provide a culture system and progenitor cell type with advantages over the current practice of BMMSC expansion on standard cell culture plastic surfaces. Cells were isolated from 6 young adult horses, expanded, and cultured on collagen-coated tissue culture plates, and no collagen control for 7 days. Samples were analyzed for cell number on days 4 and 7, and for mRNA expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin on day 7. Glycosaminoglycan (GAG) synthesis was analyzed on day 7. Differences of cell number between collagen groups and cell type, and in gene expression and GAG synthesis between collagen groups and cell types, were evaluated by use of mixed-model repeated measures ANOVA. Pair-wise comparisons were made on significant differences identified with ANOVA using Tukey's post hoc test. Statistical significance was set at P<0.05. A statistical significant (P=0.05) increase in cell number for TPCs grown on rattus collagen versus control on day 4 was observed. No difference in GAG synthesis or expression of collagen type I, collagen type III, COMP or decorin mRNA was observed between collagen groups and non-collagen controls for either cell type on day 7. TPCs cultured on all collagen types yielded more cells than similarly cultured BMMSCs on day 4, but only porcine collagen was superior on day 7. TPCs synthesized more GAG than BMMSCs when cultured on control surfaces only. BMMSCs expressed more collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and more collagen type III when cultured on control, porcine, highly-purified collagen, and rattus collagen, than TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and all collagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen when compared to BMMSCs. The results of this study revealed an advantage to culturing TPCs on randomly organized rattus collagen during the early growth phase. The beneficial effects of collagen-coated surfaces on cell proliferation is likely related to increased surface area for attachment and expansion provided by the random collagen matrix, and/or collagen-cell interactions. Tendon progenitor cells showed superior growth kinetics and expression of the matrix organizational components, COMP and decorin, than similarly cultured BMMSCs that expressed more collagen types III and I. TPCs synthesize more GAG compared to BMMSCs when cultured on plastic surfaces and there was no induction by collagen. Tendon progenitor cells should be considered as an alternative source of progenitor cells for injured equine tendons. Further in vitro studies characterizing factors that influence gene expression of both cell types is warranted. / Master of Science

Endotoxemia is a major cause of morbidity and mortality in horses. The presence of large amounts of circulating endotoxin inititates a number of cell signaling pathways leading to a systemic inflammatory response. Activation of these pathways causes the release of a number of pro- and anti-inflammatory mediators. An overwhelming release of these mediators leads to the development of clinical signs associated with endotoxemia. Treatment options are limited mostly to supportive care at this time. Mesenchymal stem cells (MSCs) have been shown to have anti-inflamamtory and immune modulatory effects that may have some benefit for the treatment of horses with endotoxemia. To evaluate the effect of equine MSCs on the response to endotoxin challenge, the study was performed on two different stem cell lines with peripheral blood mononuclear cells (PBMCs) used as controls. After stimulation with endotoxin, secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon gamma (IFN-γ) were determined by ELISA. The immunogenic properties of MSCs were assessed with a one-way mixed lymphocyte reaction. In addition, the ability of MSCs to alter production of cytokines from stimulated PBMCs was assessed. TNF-α was not produced by MSCs when compared to PBMCs (p = < 0.001). There was no significant difference between MSCs and PBMCs in the production of IL-6. IL-10 production was significantly different (p = <0.001) at 6 and 12 hours with MSCs producing more than PBMCs in one stem cell line only. MSCs did not stimulate proliferation of PBMCs. Co-incubation of MSCs with PBMCs decreased the production of TNF-α in both stem cell lines although it was not statistically significant (p = 0.4 and 0.9) at either time point. IL-6 secretion was suppressed at twelve hours with co-incubation. IL-10 production was increased with co-incubation in one stem cell line. MSCs secrete soluble factors that can alter PBMC cytokine production and they do not appear to be immunostimulatory. These findings have potential implication for treatment of equine inflammatory conditions. / Master of Science