Characterization of different subpopulations from bone marrow-derived mesenchymal stromal cells by alkaline phosphatase expression

Multiple surface markers have been utilized for the enrichment of bone marrow mesenchymal stromal cells (MSCs) and to define primitive stem cells. We classified human bone marrow-derived MSC populations according to tissue nonspecific alkaline phosphatase (TNAP) activity. TNAP expression varied among unexpanded primary MSCs, and its level was not related to colony-forming activity or putative surface markers, such as CD105 and CD29, donor age, or gender. TNAP levels were increased in larger cells, and a colony-forming unit-fibroblast assay revealed that the colony size was decreased during in vitro expansion. TNAP-positive (TNAP+) MSCs showed limited multipotential capacity, whereas TNAP-negative (TNAP-) MSCs retained the differentiation potential into 3 lineages (osteogenic-, adipogenic-, and chondrogenic differentiation). High degree of calcium mineralization and high level of osteogenic-related gene expression (osteopontin, dlx5, and cbfa1) were found in TNAP+ cells. In contrast, during chondrogenic differentiation, type II collagen was successfully induced in TNAP- cells, but not in TNAP+ cells. TNAP+ cells showed high levels of the hypertrophic markers, type X collagen and cbfa1. Mesenchymal stem cell antigen-1 (MSCA-1) is identical to TNAP. Therefore, TNAP+ cells were sorted by using antibody targeting MSCA-1. MSCA-1-positive cells sorted for TNAP+ cells exhibited low proliferation rates. Expression of cell cycle-related genes (cyclin A2, CDK2, and CDK4) and pluripotency marker genes (rex1 and nanog) were higher in TNAP- MSC than in TNAP+ MSC. Therefore, TNAP- cells can be described as more primitive bone marrow-derived cells and TNAP levels in MSCs can be used to predict chondrocyte hypertrophy or osteogenic capacity.ope