title: In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry.
abstract: During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC.
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium. catharina.deschauwer@ugent.be
authors.forename: Catharina
authors.initials: C
authors.lastname: De Schauwer
authors.forename: Sofie
authors.initials: S
authors.lastname: Piepers
authors.forename: Gerlinde R
authors.initials: GR
authors.lastname: Van de Walle
authors.forename: Kristel
authors.initials: K
authors.lastname: Demeyere
authors.forename: Maarten K
authors.initials: MK
authors.lastname: Hoogewijs
authors.forename: Jan L J
authors.initials: JL
authors.lastname: Govaere
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
authors.forename: Evelyne
authors.initials: E
authors.lastname: Meyer
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002454:Cell Differentiation; D003429:Cross Reactions; D005434:Flow Cytometry / Q000379:methods*; D006736:Horses / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008285:Major Histocompatibility Complex / Q000276:immunology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000276:immunology*; D010641:Phenotype
chemical_list: D000911:Antibodies, Monoclonal