Development and characterization of a conditionally immortalized human fetal osteoblastic cell line.
We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.
Harris
S A
SA
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.
Enger
R J
RJ
Riggs
B L
BL
Spelsberg
T C
TC
D000469:Alkaline Phosphatase / Q000378:metabolism; D000957:Antigens, Viral, Tumor / Q000235:genetics; D002113:Calcification, Physiologic; D002117:Calcitriol / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005333:Fetus / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007621:Karyotyping; D009154:Mutation / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology; D015675:Osteocalcin / Q000378:metabolism; D010641:Phenotype; D011247:Pregnancy; D013539:Simian virus 40 / Q000276:immunology; D014162:Transfection
D000957:Antigens, Viral, Tumor; D015675:Osteocalcin; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D002117:Calcitriol