Isolation and characterization of equine native MSC populations

Cristina L. Esteves, Tara A. Sheldrake, Simone P. Mesquita, Juan J. Pesántez, Timothy Menghini, Lucy Dawson, Bruno Péault, F. Xavier Donadeu

In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146(+) and CD34(+) cell populations has not been performed in any species. Immunohistochemistry was used to identify adventitial cells (CD34(+)) and pericytes (CD146(+)) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34(+) (CD34(+)/CD146(-)/CD144(-)/CD45(-)) and CD146(+) (CD146(+)/CD34(-)/CD144(-)/CD45(-)) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay. Both CD34(+) and CD146(+) cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146(+) cells were distinctly angiogenic compared with CD34(+) and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146(+) cells maintain a pericyte phenotype in culture. This study reports for the first time the successful isolation and culture of CD146(+) and CD34(+) cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146(+) cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies.