Article,
Establishing an immortalized human osteoprecursor cell line: OPC1.
J Bone Miner Res, 14 (10): 1721-33 (1999)
Abstract
The present studies evaluated the feasibility of establishing a conditionally
immortalized osteoprecursor cell line derived from human fetal bone
tissue. Primary cultures were transfected with a plasmid in which
the Mx-1 promoter drives the expression of SV40 T-antigen when activated
by human A/D interferon. Several neomycin (G418)-resistant colonies
were characterized for cell growth and alkaline phosphatase (ALP)
enzyme activity. The clone, designated OPC1 (osteoblastic precursor
cell line 1), which exhibited the highest ALP enzyme activity at
passage 10 (P10), was selected for additional osteogenic phenotypic
characterization. Reverse transcription-polymerase chain 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, OP, PTHr, and ProI mRNAs exhibited
a marked up-regulation when maintained in medium containing an osteogenic
supplement (OS). The ability to stimulate osteogenic differentiation
was characterized in postconfluent OPC1 cells maintained in tissue
culture medium supplemented with recombinant human bone morphogenetic
protein-2 (rhBMP-2) either with or without an OS. All treatment groups
exhibited a striking up-regulation of ALP enzyme activity that coincided
with ALP histochemical observations. Postconfluent cells also exhibited
the ability to form mineralized nodules under all treatments (confirmed
by von Kossa histochemical staining and calcium deposition). An enzyme
immunosorbent assay (EIA) was utilized to measure intact human OC
from the OPC1 line under the various treatments. Abundant OC was
evident in the tissue culture medium indicating de novo sythesis
and release from the OPC1 line under appropriate conditions. The
clonal human-derived OPC1 line represents a homogeneous osteogenic
cell line that not only has 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 low dose rhBMP-2
(10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor
that provides a sensitive in vitro cell culture system to evaluate
bone development, cell/biomaterial interactions, and may be a useful
screen for putative bone differentiating factors.
Tags
- ;
- alkaline
- and
- cell
- chain
- development/physiology
- division/physiology
- embryonic
- feasibility
- fetal
- histocytochemistry
- humans
- immunoenzyme
- osteoblasts/*physiology
- osteocalcin/analysis
- phenotype
- phosphatase/analysis
- polymerase
- reaction
- reverse
- studies
- survival/physiology
- techniques
- transcriptase
- transfection
