More than five decades of research in dogs has provided fundamental breakthroughs in human and veterinary medicine. Stem cell transplant has been put forward as one potential means of treating both human and canine injury; however, the use of therapeutic cellular transplantation in dogs has been hampered by a lack of knowledge of the characteristics of canine stem cells and difficulties in reproducibly isolating viable pluripotent cells of dog origin. To remedy this situation, I began a series of experiments aimed at producing induced pluripotent stem cells of canine origin (ciPSCs), comparing the properties of ciPSCs to pluripotent cell types of other species, determining the capacity of ciPSCs to give rise to multiple types of somatic tissue, and defining conditions for their continued growth. To begin, multiple primary fibroblast cell populations were derived from tissue samples taken from live adult donor dogs. After characterization of primary lines to select those with the best growth properties, fibroblasts were converted to ciPSCs by infection with high titers of recombinant retroviruses encoding the pluripotency-associated transcription factors OCT4, SOX2, c-MYC, and KLF4. Infected cultures gave rise to colonies with the characteristics of pluripotent stem cell types within several weeks, and subcloned canine iPSCs lines were found to express genes and proteins characteristic of other mammalian pluripotent cells. Like iPSCs from other species, ciPSCs were also found to have silenced expression of the viral vectors used to induce pluripotency. Clonal ciPSC displayed normal karyotypes and DNA fingerprinting analysis confirmed that iPSCs were a match for the genome of donor dogs. After a shift to culture conditions favoring differentiation, ciPSCs were observed to give rise to cells of ectodermal, mesodermal, and endodermal identity. Unlike iPSCs from many species, however, it was found that ciPSCs required the continued presence of leukemia inhibitory factor (LIF) to survive in vitro. To further elucidate the role of LIF-specific signaling pathways in maintaining ciPSC viability, we performed a series of experiments that revealed that activation of the LIF-specific JAK-STAT3 pathway was critical for preventing ciPSC death. In summary, the project performed to complete this dissertation has produced an efficient method for the derivation of pluripotent stem cells from the dog and has defined many of the molecular pathways required for their derivation and continued maintenance in vitro. This work serves as the foundation for the development of cell-based therapies for disease and injury in dogs with tremendous potential to inform our understanding of similar treatments in future human patients.
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