Changes Of Functional Activity And MiRNA Expression Profile Of Articular Cartilage Progenitor Cells In Osteoarthritis

Objective:To obtain chondrogenic progenitor cells (CPCs) from articular cartilage of patients with later stages of osteoarthritis(OA). To compare the difference of function and miRNA expression profiling between CPCs from degraded lesions and normal-appearing areas of OA articular cartilage. If possible, to establish the correlation between the abnormal function of CPCs and differentially expressed miRNA, and investigate the adjustive effect of miRNA on the function of CPCs.Method:chondrocytes from knee joint cartilage of OA patients undergoing total knee arthroplasty (TKA) were separately harvested from degraded lesions and normal-appearing areas, and then CPCs were isolated from chondrocytes using CD105and CD166co-expression markers. According to International Society for Cellular Therapy (ISCT) criteria, cell surface markers were analyzed by flow cytometry (FACS), and multi-lineage differentiation assays were performed as well to identify whether CD105/CD166expressing cells have the characteristics similar to MSCs. Subsequently, we compared the CPCs from degraded lesions with those from normal-appearing areas in terms of their percentage and proliferation rate. The potential of CPCs from two areas to differentiate to adipogenic, osteogenic, and chondrogenic lineages was observed by cytochemical staining and immunohistochemistry, and at the same time, real-time PCR was performed to detect differentiation-related genes so as to compare quantitatively their differentiation capacity. We also isolated and purified miRNA from the isolated cell subsets, and compare the difference of miRNA expression profiling between CPCs from degraded lesions and normal-appearing areas by Human miRNA OneArray chips, furthermore, significantly differentially expressed miRNAs were obtained through screening. In order to explore the adjustive effect of miRNA on CPCs, we analyzed the differentially expressed miRNAs based on Gene Ontology, and predicted the targets of miRNAs by TargetScan6.2software.Results:The selected cell subsets which express the combination of the cell-surface markers CD105and CD166were found to adhere to the culture flask, and expanded quickly in vitro. Most cells were positive for CD29, CD44, CD73, CD90, but almost negative for CD19, CD34, CD45and HLA-DR, in addition, following multi-lineage induction, they were able to differentiate to adipocytes, osteocytes and chondrocytes, what also is a requirement, together with the immunophenotype and growth pattern, to fulfill ISCT definition criteria for MSCs. The mean±SEM percentage of CD105/CD166 expressing CPCs in cartilage of degraded lesions and normal-appearing areas was0.01-22.90%/4.34-35.46%respectively, while cartilage of degraded lesions contained significantly lower numbers of CD105/CD166expressing CPCs (P=0.024<0.05) Furthermore, compared with CPCs from normal-appearing areas, the proliferation rate of CPCs from degraded lesions was significantly reduced (P=0.01<0.05). In terms of differentiation capacity, cells from degraded lesions contained less lipid vacuoles by oil red O staining accompanied with a significant lower expression of PPAR-y2mRNA, but equivalent expression of LPL mRNA during adipogenic differentiation, on the other hand, more calcium depositions and stronger alkaline phosphatase activity with a significant higher expression of Runx2mRNA were observed in cells from degraded lesions during osteogenic differentiation, and we also found both of CPCs in two areas exhibited a similar expression of Sox9, Collagen2and Aggrecan by immunohistochemistry after chondrogenic differentiation, but interestingly, a significant higher expression of Sox9and Aggrecan was detected in cells from degraded lesions. Thus, our study revealed there was a significant reduction in adipogenic potential but a significant increase in osteogenic and chondrogenic potential in CPCs from degraded lesions compared by those from normal-appearing areas. For further study,11significantly differentially expressed miRNAs was selected by miRNA OneArray chips, which included8overexpressed miRNAs (miR-574-5p, miR-30a, miR-100, miR-23b, miR-375, miR-4749-5p, miR-1273f and miR-5096) and3underexpressed miRNAs (miR-31, miR-424and miR-199a-5p). Among the above mentioned miRNAs, miR-31was the most differentially expressed miRNAs. Unfortunately, no targets of miRNAs were identified in our present study, previous studies have demonstrated that some miRNAs were associated with MSCs self-renewal and differentiation and played a key role in controlling MSCs function, such as miR-31, miR-424, miR-199a-5p, miR-100, miR-23b and miR-375. We supposed that the significant increase in osteogenic potential of CPCs from degraded lesions probably result from underexpression of miR-31. However, the real adjustive effect of miRNAs on CPCs remained to be confirmed by determination of targets and analysis of relevant signal passways.Conclusion:These findings indicate that CD105/CD166expressing cells are a group CPCs which have the similar multi-potential characteristics with MSCs. Cartilage of degraded lesions contained significantly lower numbers of CD105/CD166expressing CPCs, and the proliferative activity of CPCs from these areas is significantly reduced. CD105/CD166expressing CPCs obtained from degraded lesions hold a weaker adipogenic potential but a stronger chondrogenic and osteogenic potential, which raise the possibility that the increase in bone density and loss of cartilage as the characteristics of OA may result from turnover in the differentiation profile of CPCs that contribute to pathogenesis of OA, while miR-31maybe play a key role in this process.