308 ISOLATION, DIFFERENTIATION, AND IMMUNOPHENOTYPIC CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM EQUINE ADIPOSE TISSUE AND BONE MARROW

Abstract

Minimum criteria for the characterisation of human mesenchymal stem cells (MSC) are a) adhesion to the plastic when maintained under culture conditions; b) expression of CD105, CD73, and CD90, and no expression for CD45, CD34, and CD14; and c) differentiation into osteoblasts, adipocytes, and chondroblasts in vitro. One major difficulty in characterising equine MSC is the absence of specific monoclonal antibodies and evidence that certain markers from other species do not cross-react with the equine species. The aim of this work was to isolate, cultivate, differentiate, and conduct cellular characterisation of MSC derived from equine adipose tissue (AT) and bone marrow (BM). Adipose tissue collection was performed at the base of the horses’ tails, and BM was aspirated from the iliac crest. Mononuclear cell fraction was isolated and cultured as previously described by (Colleoni et al. 2009 Vet. Res. Commun. 33, 811–821). Chondrogenic, osteogenic, and adipogenic differentiation were performed in monolayer culture, and evidence for differentiation was made by morphological and cytological evaluations. For molecular characterisation, cells were treated with trypsin, washed with PBS, and fixed with Reagent 1 (Intraprep Kit, Beckman Coulter, Miami, FL, USA), following the manufacturer’s instructions. Samples, after washings, were incubated for 20 min at room temperature with CD105, CD90, CD44, CD45, CD34, CD14, and CD73 mAbs, directly conjugated to fluorescein isothiocyanate, PE, or APC (Beckman Coulter). Appropriate conjugate isotype controls were applied (Beckman Coulter). After staining, cells were washed twice with PBS, and fluorescence intensity was evaluated with a FC500 two-laser equipped cytometer (Beckman Coulter). Results were further analysed with the CXP dedicated program. Samples volumes were 68 ± 23.6 mL for BM and 5.6 ± 1.1 g for AT; in both AT and BM, the isolation rate was 100% (AT: 4/4; BM: 5/5). Undifferentiated cells were passaged up to 8 times for AT and 5 times for BM; population-doubling times (DT) were calculated, and data were analysed by ANOVA (Statistica for Windows, Stat Soft Inc., Tulsa, OK, USA). No significant differences (P > 0.05) were found between DT of all passages. The DT was greater (P < 0.05) for BM (3.2 ± 1.5) than for AT (1.3 ± 0.7). By passage 8, AT MSC underwent 37.3 ± 4.6 cell-doublings (CD); by passage 5, BM MSC underwent 26.2 ± 5.03 CD. Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian blue staining illustrated chondrogenesis, and positive Oil Red O staining suggested adipogenesis. The AT and BM MSC were positive for CD90, CD44, and CD105; all cell lines were negative for haematopoietic markers such as CD34, CD14, and CD45. Although marker CD73 expresses reaction in other studies involving MSC in different species, it did not cross-react with equine AT and BM MSC. Results obtained revealed the immunophenotypic characterisation of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy.