Characterization of mesenchymal stem cells derived from the equine synovial fluid and membrane

Abstract

Background: 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. 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.

Results: The 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.

Conclusion: Given 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.

Fig. 1

Cell morphology and growth. a and (b): Both cells, from the synovial fluid and the membrane, respectively, had a fibroblast-like appearance with a fusiform shape, growing in colonies. c and d: Both cultures had a growth pattern represented by a sigmoidal curve. The synovial fluid cells (SF) showed a period of adjustment without proliferation (lag phase, approximately 50 h of culture). Exponential growth was observed until 150 h, and after 168 h a decline phase was observed (c). In contrast for synovial membrane cells (SM) the lag phase was reached approximately 24 h of culture, followed by a exponential growth until 150 h and a decline phase close to 160 h (d)

Fig. 2

Expression of markers by flow cytometry on cells derived from the synovial fluid. The synovial fluid cells showed significant expression of hematopoietic (CD45, CD34, CD117, and CD133), mesenchymal (CD105 and CD90), pluripotency (Oct3/4 and Nanog), embryonic (Tra-1-81), and inflammatory (VEGF-R1 and LY6a) markers

Fig. 3

Expression of markers by flow cytometry on cells derived from the synovial membrane. The synovial membrane cells showed significant expression of hematopoietic (CD45, CD34, CD117, and CD133), mesenchymal (CD105 and CD90), pluripotency (Oct3/4 and Nanog), embryonic (Tra-1-81), and inflammatory (VEGF-R1 and LY6a) markers

Fig. 4

Expression of inflammatory and cell cycle progression markers by flow cytometry on cells derived from the synovial fluid and membrane. The TNF-r, MCP-1 and CD1a were significantly reduced in the synovial membrane cells compared to the synovial fluid cells. There was no difference in relation to the expression of the other markers (Cox-2 and CD11) between the two cultures and all markers showed significant expression. In relation to cell cycle progression markers, statistical difference between the cultures was observed only in relation to the expression of HSP47, which was increased in the synovial fluid cultures. The expression of other markers (Caspase-3, p21, Ki67, ciclin-D1, and p53) was similar between both cultures

Fig. 5

Isotype controls. a and (b) Red line corresponds to the IgG2a isotype control (mouse) and in black the specific binding in samples of synovial membrane and fluid, respectively. c and (d) Red line corresponds to the IgM isotype control (mouse) and in black the specific binding in samples of synovial membrane and fluid, respectively

Fig. 6

Analysis of the cell cycle on cells cultures derived from the synovial fluid and membrane. There were no significant differences in the proportions of cell distribution in cell cycle phases between the culture of synovial fluid (SF) cells of 48 and 144 h (a and b). In contrast, there were significant differences in the proportions of synovial membrane (SM) cells between 48 and 144 h. There was a decrease in the G2/M cell population, as demonstrated in the kinetics of cell growth and viability. After 144 h, the synovial membrane cells decreased their proliferative ability (c and d). e: The results showed a higher capacity for proliferation of synovial fluid (SF) cells between 48 and 144 h when compared to synovial membrane (SM) cells, which showed lower proliferation after 144 h in culture. The acquisitions were obtained by flow cytometer were analyzed by the Wizard Proliferation

Fig. 7

Analysis of mitochondrial electrical potential. Both synovial fluid (SF) and membrane (SM) cells cultivated after 48 and 144 h did not show differences in the electric potential. The representative histograms obtained by a flow cytometer, show the means of populations with high electric potential (MI) and low mitochondrial electrical potential (M2). In relation to the distribution of the mitochondrial number per cell, data obtained from both cultures after 48 and 144 h showed that there were a significant number of mitochondria per cell, however, without significant differences. Acquisition and analysis were performed using the WinMDI program