Isolation and characterization of human clonogenic osteoblast progenitors immunoselected from fetal bone marrow stroma using STRO-1 monoclonal antibody

Osteoprogenitor cells present in human fetal bone marrow (BM) stroma have not been characterized. We used density gradient centrifugation, aggregation on binding lectin, and enrichment by magnetic activated cell sorting with STRO-1 antibody to isolate STRO-1⁺ cells from nonadherent human fetal BM stromal cells. Immunoselected STRO-1⁺ cells were immortalized using SV-40 large T antigen and a clone, F/STRO-1⁺ A, with weak alkaline phosphatase (ALP) activity was selected. The cloned cells proliferated rapidly but were not tumorigenic. Preconfluent F/STRO-1⁺ A cells showed immunoreactivity for osteopontin, α1(I) procollagen, and parathyroid hormone-related peptide, but not for the late osteoblast differentiation markers, osteocalcin (OC), or bone sialoprotein. However, differentiation of F/STRO-1⁺ A cells was induced by dexamethasone and 1,25-dihydroxyvitamin D₃, as shown by increased ALP activity. In addition, osteogenesis occurred in F/STRO-1⁺ A cells cultured in three-dimentional aggregates, as assessed morphologically, histologically, and biochemically. Moreover, reverse transcription-polymerase chain reaction analysis showed that OC expression was silent in exponentially growing cells and occurred when cell-cell contacts were established in monolayer and in aggregates, showing induction of mature osteoblast phenotype by cell-cell contacts. Thus, clonal F/STRO- 1⁺ A cells immunoselected from human fetal BM stroma display features of immature osteoprogenitor cells which can differentiate into mature osteogenic cells by cell-cell interactions or inducing agents. The generation by immunoselection of an immortalized clonogenic human fetal BM stroma-derived cell line which behaves like an osteoprogenitor cell provides a novel model system for identifying the signals required for the commitment of osteoprogenitors in the human fetal BM stroma.