| Abstract: | Multipotent stromal cells, also termed mesenchymal stem cells or MSCs, are promising agents for cell-based bone repair. During in vitro osteogenic differentiation, human MSCs (hMSCs) readily differentiate in response to established protocols. In contrast, canine MSCs (cMSCs) respond poorly and require bone morphogenic protein-2 (BMP-2) for consistent osteogenic differentiation. The goal of the present study was to comprehensively assess the transcriptional activity of cMSCs and hMSCs during in vitro osteogenic differentiation, allowing for the identification of important similarities and differences in osteogenic differentiation pathways. Through the use of RNA sequencing (RNA-Seq), a comprehensive analysis of transcriptomes was performed, facilitating a detailed evaluation of crucial osteogenic pathways. We hypothesized that culture-expanded cMSCs failed to initiate autocrine BMP-2 production as compared to hMSCs. Osteogenesis of representative bone-marrow-derived cMSCs and hMSCs was assessed under three conditions: control, traditional osteogenic differentiation medium (ODM), and osteogenic differentiation medium supplemented with 200 ng/mL of recombinant human BMP-2 (ODM+BMP-2). Early-stage osteogenic differentiation was evaluated using alkaline phosphatase (ALP) activity with late-stage differentiation assessed via Alizarin Red staining. RNA-Seq was performed at days 0, 7, 14, and 21. Canine MSCs had weak ALP activity without BMP-2 supplementation, whereas hMSCs exhibit strong ALP activity in all conditions. Mineralization was documented in canine ODM+BMP-2 and both human osteogenic conditions. Through the use of RNA-Seq we identified for the first time that cMSCs are capable of producing endogenous canine BMP-2. BMP-2 expression was significantly upregulated in cMSCs with BMP-2 supplementation during mid-to-late stage osteogenic differentiation (Days 14, 21) and cMSCs without exogenous BMP-2 supplementation (ODM) had a downregulation of expression at critical early timepoints (Days 7, 14). When compared to cMSCs, human MSCs primarily had a downregulation of BMP-2 across all timepoints in ODM, the classic medium for inducing hMSCs. Finally, canine MSCs exhibited a higher differential expression of BMP antagonists as compared to hMSCs. Classical downstream markers of osteogenesis were identified in both MSCs. In conclusion, canine and human MSCs regulate BMP-2 expression differently during in vitro osteogenesis. This study represents a critical advancement for human and canine musculoskeletal tissue engineering and will provide foundational data for future translational studies. The electronic version of this dissertation is accessible from https://hdl.handle.net/1969.1/198673 |
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