In most human primary bone cells, SV40 T-antigen expression was able to expand life span for a few passages before cells undergo growth arrest, described as crisis. In this study, telomerase activity was reconstituted in human osteoblast precursors (hPOB cells) and marrow stromal cells (Saka cells) transformed with the SV40 T antigen. Bone cells with telomerase activity were able to bypass crisis and show unlimited life span. Despite chromosomal aberrations observed in hPOB-tert cells, these immortalized precursors were able to differentiate into osteoblasts like precrisis hPOB cells. Saka-tert cells enhanced the formation of human osteoclast-like cells in a similar manner as Saka cells. These results demonstrate that reconstitution of telomerase activity in transformed SV40 T-antigen human osteoblast precursors or marrow stromal cells leads to the generation of immortalized cells with a preserved phenotype.

PURPOSE:To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies.METHODS:Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays.RESULTS:The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase.CONCLUSIONS:The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.

Human liver tissue slices, homogenates, organ cultures, subcellular fractions and primary hepatocyte cultures represent valuable tools for analysis of metabolism, toxicity and efficacy of xenobiotics. However, the limited access to human tissue and the high test variability with different tissue isolates underline the significance of having standardized and defined human liver cell models. This report describes the establishment of replicative cultures of human liver epithelial cells and their immortalization in serum-free medium. Highly efficient immortalization is achieved by the infection of primary liver cells with a retroviral recombinant carrying the simian virus 40 (SV40) large T antigen gene. These cultures have an indefinite life-span, are non-tumorigenic and express differentiation markers and metabolic functions of normal primary hepatocytes. Therefore, these cells are useful for various pharmaco-toxicological applications.