Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment

Research Open Access Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment Joice Fülber1, Durvanei A. Maria2, Luis Cláudio Lopes Correia da Silva3, Cristina O. Massoco4, Fernanda Agreste1 and Raquel Y. Arantes Baccarin1Email authorView ORCID ID profile Stem Cell Research & Therapy20167:35 https://doi.org/10.1186/s13287-016-0294-3 ©  Fülber et al. 2016 Received: 29 May 2015Accepted: 17 February 2016Published: 5 March 2016 Abstract Background Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. Methods Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-Cnu/nu mice to verify the safety of the MSCs from these sources. Results Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects. Conclusions SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity. Keywords EquineMesenchymal stem cellSynovial fluidSynovial membraneAllogeneic cell bank Background Osteoarticular diseases have received a substantial amount of scientific attention in recent years, primarily because of their high prevalence and significant impact on the equine industry. These diseases irreparably damage articular cartilage and negatively influence athletic performance in horses. Proper treatment has therefore been sought to facilitate the regeneration of hyaline cartilage and to maintain the integrity of its structure. For this purpose, the use of cellular therapies, including mesenchymal stem cells (MSCs) from various sources, is a promising tool for the treatment of osteoarticular disease. MSCs are characterized by their proliferative ability and their capacity to differentiate into several mesenchymal lineages, such as osteoblasts, chondrocytes, adipocytes, tenocytes, and myocytes; therefore, they are classified as multipotent progenitor cells [1–3]. Regenerative medicine provides an opportunity to control the evolution of the disease due to the immunomodulatory, anti-inflammatory, and tissue regenerative properties of MSCs. In this context, the use of appropriate populations appears to be crucial for the successful regeneration of damaged articular structures [4]. Regarding horses, MSCs have been obtained from bone marrow, adipose tissue [5], umbilical cord [6–8], umbilical cord blood [9], amniotic membrane [10], peripheral blood [11], and recently from synovial fluid (SF) and synovial membrane (SM) [12, 13]. Although MSCs from synovial tissues have abilities comparable with those of MSCs from other sources, they have also been shown to possess high chondrogenic potential. Additionally, it was inferred that these cells are already predisposed to differentiate into chondrocytes, suggesting that the ancestral microenvironment directs the “destination” cell upon differentiation [14]. These observations support the hypothesis that these cells may be prime candidates for the regeneration of cartilage [15–18]. SM collection can be performed during arthroscopy [4, 19–22], and SF can easily be collected through arthrocentesis [23]. Although autologous therapy with MSCs does not result in any deleterious effects, its use in horses still has limitations, such as the inability to initiate treatment immediately after arthroscopic diagnosis because the expansion of MSCs in culture takes 15–26 days [3]. The treatment of older horses is also limited because there is an apparent decrease in the abilities of MSCs in this population. Allogeneic treatment eliminates the long timeframe that is required to isolate and expand MSCs. Typically, SF or SM is harvested in cases of osteoarthritis (OA) or osteochondritis dissecans (OCD) during an arthroscopic procedure that is being conducted for prognostic purposes, and these samples can also be used to create a cell bank for allogeneic therapy. However, it is not currently known whether these cell sources exhibit characteristics similar to those of cells from healthy joint tissues. Even in the case of allogeneic therapy, the harvest of synovial tissue during arthroscopic treatments of joints with OA or OCD may not the best choice, as SF or SM could instead be harvested from contralateral healthy joints. However, there have been few reports of the biological characterization of equine MSCs from synovial tissues, so concomitant quantitative and qualitative assessment should therefore be encouraged to identify these synovial-derived cells and to provide additional information about them. Based on this research scenario, we outlined a study to compare the phenotype, morphology, and multilineage differential potential of MSCs from synovial fluid (SF-MSCs) and from synovial membrane (SM-MSCs) of horses, using healthy joints, joints with OA, and joints with OCD. Further, to verify that these SF-MSCs and SM-MSCs would not differentiate into tumoral cells, we used a mouse tumorigenicity test. Methods Animal ethics This s (...truncated)