Establishing an immortalized human osteoprecursor cell line: OPC1

The present studies evaluated the feasibility of establishing a conditional ly immortalized osteoprecursor cell line derived from human fetal bone tiss ue. Primary cultures were transfected with a plasmid in which the Mx-l prom oter drives the expression of SV40 T-antigen when activated by human AID in terferon, Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, des ignated OPC1 (osteoblastic precursor cell line 1), which exhibited the high est ALP enzyme activity at passage 10 (P10), was selected for additional os teogenic phenotypic characterization. Reverse transcription-polymerase chai n reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC) , osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OF, PTHr, and ProI mRNAs exhibited a marked up-regul ation when maintained in medium containing an osteogenic supplement (OS), T he ability to stimulate osteogenic differentiation was characterized in pos tconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or wi thout an OS, All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postco nfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium d eposition). An enzyme immunosorbent assay (EIA) was utilized to measure int act human OC from the OPC1 line under the various treatments. Abundant OC w as evident in the tissue culture medium indicating de novo sythesis and rel ease from the OPC1 line under appropriate conditions. The clonal human-deri ved OPC1 line represents a homogeneous osteogenic cell line that not only h as maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of tow dose rhBMP-2 (10 ng/ml), Thus, the OPC1 line is a human-der ived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a u seful screen for putative bone differentiating factors.