Detection of Cell Proliferation and Apoptosis in Early Osteoblast Lineage

Primary calvarial osteoblast culture is widely used to examine the progression of the osteoblast lineage. Due to the heterogeneous nature of cell populations, it is difficult to precisely assess the progression of different subpopulations in culture. Objectives: The objective of this study is to demonstrate the utility of a fluorescence-marked culture combined with FACS analysis to examine cell proliferation and apoptosis in different osteoblastic subpopulations. Methods: Transgenic mice harboring green fluorescent protein (GFP) driven by the type I collagen promoter (pOBCol3.6GFP) were used to establish primary calvarial osteoblast cultures. Proliferating cells were labeled with BrdU and detected by a fluorescent APC-conjugated anti-BrdU. Apoptosis was detected by annexin V, which was tagged with fluorescent APC. In FACS analysis, the GFP(+) and GFP(-) subpopulations were analyzed for the composition of cells in the cell cycle and apoptosis. To test the present analysis, parathyroid hormone (PTH) was added to cultures to determine its effect on cell proliferation and apoptosis. Results: In early calvarial osteoblast cultures, there was 4- to 5-fold more cell proliferation and apoptosis at day 3 than at day 7. Although GFP(+) and GFP(-) cells had similar rates of cell proliferation, there were more GFP(+) cells in the G2+M phase and more GFP(-) cells in the G0/G1 phase. PTH treatment resulted in a 10-15% decrease in cell proliferation and this effect was mostly due to the delay of cells in the G0/G1 phase. In contrast to a similar proliferation rate between GFP(+) and GFP(-) cells, there was a higher proportion of GFP(-) cells in apoptosis. Apoptosis was decreased in PTH-treated cultures and this effect was more obvious in the GFP(-) subpopulation. Conclusions: The present analysis demonstrated the advantage of using fluorescent makers to examine cell differentiation and apoptosis in the heterogeneous osteoblast cultures. Supported by NIH K12HD01409 and R03DE015224.