Journal of Cellular Biochemistry 87:9–15 (2002) ARTICLES Further Characterization of Human Fetal Osteoblastic hFOB 1.19 and hFOB/ERa Cells: Bone Formation In Vivo and Karyotype Analysis Using Multicolor Fluorescent In Situ Hybridization M. Subramaniam, 1 * Syed M. Jalal, 2 David J. Rickard, 1 Steven A. Harris, 3 Mark E. Bolander, 4 and Thomas C. Spelsberg 1 1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905 2 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 3 Bayer Corporation, West Haven, Connecticut 06516 4 Department of Orthopedics, Mayo Clinic, Rochester, Minnesota 55905 Abstract We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178–1860). To char- acterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1 – 2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2 – 3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro. J. Cell. Biochem. 87: 9–15, 2002. ß 2002 Wiley-Liss, Inc. Key words: osteoblasts; differentiation; hFOB cells; hFOB/ER cells; MG63 cells; karyotype analyses; multiprobe FISH; in vivo bone formation; matrix production The availability of good model cell lines for studies of human osteoblast function, differ- entiation, regulation by hormones, and mech- anical stress, etc., have been limited. Of the osteoblast model systems that have been estab- lished to characterize osteoblast growth and differentiation in vitro, the two that are widely used are primary cultures obtained from human bone fragments and osteosarcoma derived cell lines generated from human bone tumors. The primary osteoblast cultures are an excellent normal model system but they have their limi- tations due to heterogeneity of phenotype and stage of differentiation, a slow growth rate, and limited life span in culture. Several transform- ed human osteosarcoma cell lines are used as alternatives to primary cultures, including MG63 line [Heremans et al., 1978], SaOS-2 [Fogh et al., 1977], U2-OS [Poten and Saksela, 1967], and TE-85 cells [McAllister et al., 1971]. Although these cell lines are more homogenous, they usually do not exhibit the complete pheno- type of differentiated osteoblasts, have abnor- mal growth properties including loss of cell contact inhibition, and exhibit responses to hormones and cytokines that sometimes differ ß 2002 Wiley-Liss, Inc. Grant sponsor: National Institutes of Health; Grant numbers: AG04875, DE14036; Grant sponsor: Mayo Foun- dation; Grant sponsor: NIH (to D.K. as training grant); Grant number: DK07352. *Correspondence to: M. Subramaniam, Department of Bio- chemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905. E-mail: subramaniam@mayo.edu Received 22 May 2002; Accepted 24 May 2002 DOI 10.1002/jcb.10259