Validation of multiparametric panels for bovine mesenchymal stromal cell phenotyping

Bovine mesenchymal stromal cells (MSCs) display important features that render them valuable for cell therapy and tissue engineering strategies, such as self-renewal, multi-lineage differentiation, as well as immunomodulatory properties. These cells are also promising candidates to produce cultured meat. For all these applications, it is imperative to unequivocally identify this cell population. The isolation and in vitro tri-lineage differentiation of bovine MSCs is already described, but data on their immunophenotypic characterization is not yet complete. The currently limited availability of monoclonal antibodies (mAbs) specific for bovine MSC markers strongly hampers this research. Following the minimal criteria defined for human MSCs, bovine MSCs should express CD73, CD90, and CD105 and lack expression of CD14 or CD11b, CD34, CD45, CD79α, or CD19, and MHC-II. Additional surface proteins which have been reported to be expressed include CD29, CD44, and CD106. In this study, we aimed to immunophenotype bovine adipose tissue (AT)-derived MSCs using multi-color flow cytometry. To this end, 13 commercial Abs were screened for recognizing bovine epitopes using the appropriate positive controls. Using flow cytometry and immunofluorescence microscopy, cross-reactivity was confirmed for CD34, CD73, CD79α, and CD90. Unfortunately, none of the evaluated CD105 and CD106 Abs cross-reacted with bovine cells. Subsequently, AT-derived bovine MSCs were characterized using multi-color flow cytometry based on their expression of nine markers. Bovine MSCs clearly expressed CD29 and CD44, and lacked expression of CD14, CD45, CD73, CD79α, and MHCII, while a variable expression was observed for CD34 and CD90. In addition, the mRNA transcription level of different markers was analyzed using reverse transcription quantitative polymerase chain reaction. Using these panels, bovine MSCs can be properly immunophenotyped which allows a better characterization of this heterogenous cell population.