Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.