The Effects of Chemical Reagents and Physical Environment on the In Vitro and In Vivo Properties of Adipose-Derived Multipotent Stromal Cells Isolated from Different Species

Adult adipose derived mesenchymal stromal cells (ASCs) have been characterized in various species. Many factors may affect ASC fate and ASCs from different species may have different response to these factors. The first study was to identify the differences of the canine ASCs isolated from subcutaneous and infrapatellar adipose tissues, and evaluate the impact of cryopreservation on the cells. Based on paired comparisons of fresh and cryopreserved ASCs, cryopreserved ASCs had lower CD29 and CD44 protein expression and lower proliferation rates. The cryopreserved ASCs had relative lower mitochondria in the cytoplasm compared to the fresh ASCs regardless of tissue sources. The second study was to apply human ASCs for bone regeneration. The spinner flask bioreactor system was employed to load human ASCs onto three commercial scaffolds and the cell-scaffold constructs were cultured in stromal, osteogenic, or osteogenic for 48 hours followed by stromal medium for up to 28 days. The distinct scaffold upregulated different osteogenic signaling pathways, suggesting distinct osteogenic cell signaling pathways were selectively upregulated by scaffold composition. The third study was designed to quantify in vivo equine multipotent stromal cell (MSC) osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Addition of mineral to polymer scaffolds enhanced equine MSC osteogenesis over polymer alone, and contributions by both exo- and endogenous MSCs were confirmed. The fourth study was designed to evaluate the effects of collagenase digestion and cryopreservation on equine ASCs. Higher collagenase concentration yielded more nucleated cells, and the percentages of MHCII-, CD44+, CD105+ cells in freshly isolated and cryopreserved cells were similar. The embryonic gene expression was enhanced and the essential gene expression decreased after cryopreservation. The fifth study was to demonstrate the endodermal transdifferentiation capability in feline ASCs. xvi Feline-specific pancreatic β cell induction medium was developed in the study, and islet-like cell clusters that secrete insulin in response to glucose stimulation were created. Overall, the investigations in this dissertation provide critical information for canine, feline, equine and human MSC-based tissue engineering therapies and may contribute to better efficiency and efficacy of cel