384 ISOLATION OF MESENCHYMAL STEM CELLS FROM WHARTON'S JELLY, CORD BLOOD, AND AMNIOTIC FLUID IN THE HORSE

The effect of orthopedic injuries on the health of racehorse and racing financials can be dramatic. Bearing this in mind, there is naturally a great interest in new possible treatments for orthopedic injuries. In this study we evaluated equine amniotic fluid (AF), cord blood (CB), and Wharton’s jelly (WJ) as alternative sources of primitive, multipotent stem cells. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Samples were recovered at labor from 7 standardbred mares with ages between 6 and 15 years. Wharton’s jelly was incubated with collagenasis at 37°C for at least 2 hr. The resulting cell suspension was centrifuged, and the cell pellet was resuspended in culture medium (DMEM and TCM-199, 1:1) plus 10% FBS (GIBCO®, Invitrogen Corporation, Carlsbad, CA, USA), 100 IU mL-1 penicillin, and 100 μg mL-1 streptomycin). Amniotic fluid and CB were diluted 1:1 in PBS medium supplemented with antibiotic solution and centrifuged for 15 min at 300 x g. The pellet was resuspended in 5 mL of culture medium, placed on 5 mL of 70% Percoll solution, and centrifuged at 1200 x g for 30 min. The interface layer was then aspirated and resuspended in culture medium. All the cell lineages were washed seeded into 25 cm2 flasks and cultured in a 5% CO2 incubator at 38.5°C. Medium was refreshed after 48 h and then twice a week. Adherent fibroblast-like cells were tested for their differentiation potential at passage 3. Chondrogenic differentiation was performed using a micromass culture technique, whereas osteogenic differentiation was induced in monolayer culture (Mizuno H and Hyakusoku H 2003 J. Nippon Med. Sch. 70, 300-306). Evidence for differentiation was made via morphological, biochemical, and cytological evaluations. Sample volume was 15-55 mL for AF, 10-60 mL for CB, and 5-12.6 g for WJ. In 4 out of 6 (66.7%) AF, 5 out of 5 (100%) CB, and 6 out of 7 (85.7%) WJ samples, colonies with the classical mesenchymal stem cell (MSC) morphology of adherent fibroblastoid spindle-shaped cells growing in a monolayer were observed. Undifferentiated cells have been passaged up to 8 times, population-doubling times (DT) were calculated for a subset of cell passages, and data were analyzed by ANOVA (Statistica for Windows, Stat Soft Inc., Tulsa, OK, USA). In all samples, there were no significant differences (P > 0.05) between DT of all passages. The DT was greater (P < 0.05) forCB (2.6 ± 1.3 d) than for AF (2.1 ± 0.8 d) and WJ (1.9 ± 0.5 d). By passage 8, the cells had undergone 39.1 ± 1.2 cell-doubling numbers in AF, 35.1 ± 2.7 in CB, and 37.4 ± 2.0 in WJ. A spheroid structure and positive Alcian blue staining of matrix glycosaminoglycans illustrated chondrogenesis. Cellular morphology and positive von Kossa staining, as well as alkaline phosphatase activity, confirmed osteogenesis. Based on the results obtained, WJ, CB, and for the first time in the horse, AF represent alternative sources of stem cells. Their differentiation into osteocytes and chondrocytes demonstrated that equine MSC from these samples can be induced to form multiple cell types, which underlies their value for regenerative medicine in injured horses.