Chondrogenic capacities of equine synovial progenitor populations

Articular Cartilage Repair;Stem Cells;Synovium-derived Stem Cells;bone morphogenetic protein 2 (BMP-2)

Chen, Yu-Wen

关键词: Articular Cartilage Repair;    Stem Cells;    Synovium-derived Stem Cells;    bone morphogenetic protein 2 (BMP-2);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34334/CHEN_YU-WEN.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Osteoarthritis is linked to approximately 60% of equine lameness problems. This disease involves compromised homeostasis of articular cartilage and eventual loss of this tissue in affected joints. Mesenchymal stem cells (MSCs) provide promise for cell-based therapies for articular cartilage repair; however, MSCs from specific tissues exhibit source-based differences in phenotypic and biosynthetic activities, influencing the utility of specific MSC populations for clinical applications.The objective of this research program is to provide strategies using synovial progenitor populations for articular cartilage repair in horses.The synovial fluid samples were added directly to culture medium and maintained until confluence. The primary cultures were passage twice to generate sufficient cell numbers for differentiation. Monolayer cells were treated with osteogenic and adipogenic medium for 14 days and confirmed by cytochemical analysis.For chondrogenesis, cells were maintained in pellet culture with chondrogenic medium for 20 days. Expression of chondrocytic phenotypes was assessed by quantitative RT-PCR of chondrocyte-specific genes, DMMB assays for deposition of sulphated glycosaminoglycans (sGAG), an ELISA assay for collagen type II (Coll II) deposition and the alkaline phosphatase (ALP) bio-assay. The results showed that equine synovial fluid-derive MSCs is an alternative cell source for articular cartilage repair and might be more appropriate source than MSCs isolated from bone marrow.To compare the chondrogenic capacities of MSCs isolated from equine synovium (SYN), bone marrow (BM) and adipose (FAT) tissue, six healthy adult horses were used in this study. BM aspirates were collected from tuber coxae, SYN tissues were collected from right radiocarpal joint, and FAT tissues were collected from the subcutaneous space adjacent the tail. Synovium and fat were minced and digested in 0.2% collagenase for 3 hr. Primary cell isolates were seeded at low density and expanded through two passages. After expansion, cells were transferred to pellet cultures in DMEM with ITS, ascorbic acid, sodium pyruvate, dexamethasone and TGF-β1 for 20 days. Chondrogenesis was assessed by Q-PCR of chondrocytic gene expression, measurement of Col II and sGAG secretion, and by ALP activity. The results showed that SYN cells are phenotypically more appropriate for articular chondrogenesis than MSCs from bone marrow or fat.We also have developed equine BMP-2 adenoviral expression system that can induce chondrogenesis in equine synovium-derived cells. The putative equine BMP-2 sequence was generated by blasting the human and murine BMP-2 cDNA sequences across the equine genome data base. The open reading frame of equine BMP-2 was obtained from equine articular chondrocyte RNA by gene specific RT-PCR, following by two rounds of nested PCR amplification. The nested PCR product was sub-cloned into pCMV-SPORT to generate the eqBMP-2 expression vector and into the VQAd CMV K-NpA shuttle plasmid to generate the equine BMP-2 adenoviral expression system. The results showed that the mature eqBMP-2 protein contained 114 amino acids and was completely consistent with the amino acid sequences of the mature human and murine BMP-2 proteins, and the adenovirus, when applied to cells at an MOI of 100, generated BMP signaling activity equivalent to approximately 25 ng BMP-2/ml. The eq-BMP2-infected cells deposited significantly higher sGAG and formed larger size of pellets than did control and LacZ-infected groups.