Abstract
BackgroundIsolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were characterized by morphology and expression of specific markers for stem cells, pluripotency, inflammation, and cell cycle.ResultsThe medium MEM was more effective (97 % ± 2) to maintain both cultures. The cultures were composed by adherent cells with fibroblast-like shape, which had a growth pattern represented by a sigmoidal curve. After the expansion, the cells were analyzed by flow cytometry for stem cells, inflammatory, and cell cycle markers, and both lineages showed significant expression of CD45, Oct3/4, Nanog, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF, and LY6a. In contrast, there were differences in the cell cycle phases between the lineages, which was not observed in relation to the mitochondrial electrical potential.ConclusionGiven the large impact that joint pathology has on the athletic performance horses, our results suggested that the SF and SM are promising sources of stem cells with satisfactory characteristics of growth and gene expression that can be used in equine regenerative medicine.
Highlights
Isolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac
Morphology and culture growth In order to standardize the best culture medium to maintain the synovial fluid and membrane, 4 tests were performed using 4 different types of mediums supplemented with 10 % of fetal bovine serum (FBS) and 1 % of antibiotics - penicillin and streptomycin (Table 1)
After the culture expansion, a loss of cell adhesion in the substrate and development of multi-cell aggregates in the culture, resulting in cell death, was observed (Table 1). Both cells from the synovial fluid and the membrane had a fibroblast-like appearance with a fusiform shape (Fig. 1a and b) that was maintained after freezing
Summary
Isolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses In equines, these cells have being considered promising candidates for articular regeneration. Bone marrow is considered a good and acceptable source of stem cells, the synovial membrane and its fluid are tissue-specific, which leads to a chondrogenic and expansion potential greater than other sources. These cells can be obtained by minimally invasive techniques [6,7,8,9]. Considering that treating osteoarthritis, which causes persistent pain and contributes to chronic lameness, is difficult in chronic diseases, with a reserved prognosis [11,12,13], and the growing interest for this field especially in regard to the search for new strategies for treatment, we are establishing a protocol to culture and characterize mesenchymal stem cells from equine synovial fluid and from the synovial membrane, which in the future can be used to treat osteoarthritis, especially when surgical intervention is not viable
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