Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells.

Osteopontin (OPN), a major component of the bone matrix, is expressed at different stages of bone formation. To determine possible relationships between OPN expression and stages of osteogenic cell differentiation, we have performed single cell analyses of intracellular OPN in early (proliferating), subconfluent (differentiating), and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC) using a combination of flow cytometry and confocal microscopy. At each culture stage, a high proportion (60-98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a small proportion of OPN-ve cells which were characterized by their small size, low granularity, high proliferative capacity, and enhanced osteogenic potential. The OPN+ve cells displayed two distinct patterns of intracellular immunostaining: a perinuclear distribution typical of secreted proteins and a perimembrane distribution in which patches of OPN were concentrated at the cell surface. Perimembranous staining predominated in migrant cells, which contained greater than tenfold higher levels of OPN than nonmigrant cells as separated in a Boyden chamber. When cell proliferation was high (day 2), most cells were OPN + ve. At all culture stages the intensity of OPN staining was increased as cells progressed through the cell cycle. As cells differentiated and started to form matrix (days 4 and 6), the mean cell expression of OPN was also increased (fourfold), independent of changes in total cell protein. However, despite the association of OPN with osteogenic cells, we were surprised to find that a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these cell populations was confirmed by RT-PCR, and a strong correlation was observed between the quantitative flow cytometry data and Western blot analysis of cell extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies, therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: small OPN-ve cell populations enriched in osteogenic precursors, differentiating osteogenic cells that synthesize and secrete OPN, and migrating stromal cells characterized by a perimembranous OPN staining pattern.