Temporal Analysis of Equine Bone Marrow Aspirate During Establishment of Putative Mesenchymal Progenitor Cell Populations

Abstract

Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB cell surface molecules. After 2 h of culture, bone marrow mononuclear cells had a phenotype of CD44(hi), CD29(hi), CD90(lo), CD11a/CD18(hi), and CD45RB(lo). Isolated mononuclear cells were analyzed by flow cytometry and RT-qPCR at 2, 7, 14, 21, and 30 days of culture. At all culture time points, gene expression was in agreement with cell surface protein expression. In established cultures of MPCs, cells remained robustly positive for CD44 and CD29. The proportion of positive cells and the mean fluorescence intensity of positive cells increased in CD90 expression as MPC cultures became more homogeneous. Inversely, the population of cells in culture decreased expression of CD11a/CD18 and CD45RB molecules over time. The decreased expression of the latter molecules makes these useful negative markers of established MPC cultures under normal expansion conditions. The results of this study demonstrate numerous dynamic changes in cell surface molecule expression during early establishment of MPC populations, which may aid to improve MPC isolation methods for research or therapeutic applications.