Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors.

Abstract

Human bone marrow contains a distinct cell population that expresses bone proteins and responds to transforming growth factor p1 (TGF-,B), but not to hematopoietic growth factors (Long, M. W., J. L. Williams, and K. G. Mann. 1990. J. Clin. Invest. 86:1387-1395. We now report the isolation, characterization, and growth factor responsiveness of these precursors to human osteoblasts and the identification of a human osteoprogenitor cell. Immunological separation of human bone marrow nonadherent low-density (NALD) cells results in a marked enrichment of cells that express osteocalcin, osteonectin, and bone alkaline phosphatase. Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone antigen-positive cells are approximately the size of a lymphocyte, whereas other, less frequent antibody-separated subpopulations consist of osteoblast-like cells and osteoprogenitor cells. In serum-free cultures, TGF-.8 stimulates the small, antigen-positive cells to become osteoblastlike, as these cells both increase in size, and express increased levels of osteocalcin and alkaline phosphatase. Antibody-separated cells also contain a separate population of clonal progenitor cells that form colonies of osteoblast-like cells when cultured in serum-free, semi-solid media. Two types of human osteoprogenitor cells are observed: a colonyforming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells. Osteopoietic colony-forming cells and cluster-forming cells have an obligate but differential requirement for osteogenic growth factors. The CFCs respond to TGF-,B, basic fibroblast growth factor (bFGF), bone morphogenic protein-2 (BMP-2), and 1, 25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominately by 1,25-OH D3 and TGF-fi, but fail to respond to bFGF. We conclude that human bone marrow Clin. Invest. 1995. 95:881-887.)

Characterization of circulating osteoblast lineage cells in humans

“…Moreover, molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these cell types. These findings thus lent considerable credence to the previous work of Long et al [3,4] and established that, at least in the experimental paradigm used by Dominici and colleagues [9], non-adherent bone marrow cells have a >10-fold more robust bone-repopulating activity than do adherent bone marrow stromal cells. Moreover, the findings were also consistent with previous work by Olmsted-Davis et al [10] suggesting the presence of a unique progenitor cell with both hematopoietic and osteoblastic differentiation potential in the non-adherent subset of bone marrow cells.…”

“…We found that OCN pos cells isolated using either the antibody we used in our previous work (SC V-19) [14] or the antibody used by Long and colleagues (HT AON-5031) [3,4] identified very similar populations of cells, at least as assessed by flow cytometry and confocal microscopy. While additional studies need to be done further characterizing the cells isolated with the two antibodies, these data do provide some reassurance that the cells identified in our previous work using the SC V-19 antibody are not some type of spurious population staining only with this particular antibody.…”

“…We initially used the anti-OCN antibody utilized in our previous study (SC V-19) [14] and the antibody originally used by Long and colleagues in their work for identifying OCN pos cells in bone marrow (HT AON-5031) [3,4] and verified that both antibodies stained only mineralized matrix and the appropriate cells in normal human bone ( Figure 1A and B). Adjacent muscle or connective tissue of the same section did not stain with the two OCN antibodies ( Figure 1C and D) Human bone incubated just with the secondary antibody served as negative control ( Figure 1E and F).…”

“…Since then, there has been an extensive body of work (summarized in [2]) on the characterization of bone marrow stromal cells with osteoblastic potential in rodent and in human systems. Concurrent with this work, however, Long and colleagues identified, over a decade ago, a non-adherent population of cells in bone marrow with osteogenic potential [3,4]. Rather than selection by adherence to plastic, the primary assay used was cell sorting using antibodies to osteocalcin (OCN), osteonectin, and bone alkaline phosphatase (AP).…”

“…Rather than selection by adherence to plastic, the primary assay used was cell sorting using antibodies to osteocalcin (OCN), osteonectin, and bone alkaline phosphatase (AP). When cultured in the presence of TGF-β and accessory bone marrow cells (the identity of which still remains unclear), OCN positive (OCN pos ) cells proliferated and differentiated into mature osteoblastic cells expressing bone-related genes and capable of mineral deposition [3,4]. Somewhat surprisingly, despite the fact that OCN is a secreted protein, these investigators did not need to permeabilize the cells in order to detect cells producing OCN.…”