title: Immunophenotypic characterization of ovine mesenchymal stem cells.
abstract: The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, North Mymms, Hatfield, Herts, AL9 7TA, United Kingdom.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Khan
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, North Mymms, Hatfield, Herts, AL9 7TA, United Kingdom.
authors.forename: Anil
authors.initials: A
authors.lastname: Chandrashekran
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, North Mymms, Hatfield, Herts, AL9 7TA, United Kingdom.
authors.forename: Roger K W
authors.initials: RK
authors.lastname: Smith
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, North Mymms, Hatfield, Herts, AL9 7TA, United Kingdom.
authors.forename: Jayesh
authors.initials: J
authors.lastname: Dudhia
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D002454:Cell Differentiation; D019902:Chondrocytes / Q000166:cytology / Q000276:immunology; D017364:Collagenases / Q000737:chemistry; D003429:Cross Reactions; D004492:Edetic Acid / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology; D010006:Osteoblasts / Q000166:cytology / Q000276:immunology; D010447:Peptide Hydrolases / Q000737:chemistry; D061251:Primary Cell Culture; D012377:Rodentia; D012756:Sheep
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015415:Biomarkers; C575294:accutase; D004492:Edetic Acid; D010447:Peptide Hydrolases; D017364:Collagenases
keywords: bone marrow; immunophenotype; mesenchymal stem cell; ovine