The Characterization of Bovine Adipose-Derived Stem Cells in Conventional and Co-culture Environments for Tissue Engineering
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Adipose-derived stem cells (ASCs) have been extensively investigated for their applicability in the field of tissue engineering due to their multi-lineage differentiation potential and the convenience of cell acquisition. To date, conventional inductive media have been used to induce lineage-specific differentiation of ASCs; however, this general approach has limitations in terms of high costs and unstable differentiation responses. In native tissues, mesenchymal stem cells (MSCs) interact with their surrounding cells (i.e. mature cells) through paracrine and autocrine signaling, which can regulate their metabolism and cell function. Therefore, the author developed in vitro co-culture models to study the interactions between ASCs and three different mature cell types: adipocytes, chondrocytes and osteoblasts. In this work, bovine ASCs (bASCs) from the interdigital fat pad were first isolated and characterized, in terms of in vitro proliferation and multi-lineage (bone, cartilage, fat) differentiation with conventional inductive media and culture conditions. Doubling time calculations and gene expression analysis of stem cell markers indicated a threshold existed for stem cell degeneration at passage 5 (P5) for bASCs when expanded extensively in vitro. The multi-lineage differentiation potentials were compared between passage 2 (P2) and P5. Interestingly, while the P5 bASCs presented significantly lower levels of adipogenesis and chondrogenesis, osteogenesis was maintained or even improved with serial passaging. In the designed indirect co-culture systems, adipogenesis and chondrogenesis were investigated in growth medium without key differentiation factors, whereas osteogenesis was induced in conventional osteogenic medium, to maintain the stable phenotype of the mature osteoblasts in culture. The results were used to demonstrate the general feasibility of mature cell-induced or -enhanced bASC differentiation through soluble, cell-secreted paracrine signaling. When compared to growth factor (GF)-stimulated differentiation, the bASCs in co-culture presented an earlier, but potentially stronger, level of differentiation. Among these paracrine factors, Wnt proteins are known to play essential roles in mediating stem cell self-regulation and fate determination. In the current thesis, the Wnt inhibitors WIF-1 and DKK-1 were used to explore the involvement of the Wnt canonical and non-canonical signaling pathways in the designed co-culture environments. The data showed a strong correlation with the literature, indicating the canonical pathway was upregulated during osteogenesis, but inhibited during adipogenesis. The inhibition of chondrogenesis through the canonical pathway was suggested on a genetic level.