Telomerase Immortalized Cells Have Stem Cell Characteristics

J.W. Shay
University of Texas Southwestern Medical Center, Dallas, TX

Ectopic expression of human telomerase (hTERT) produces telomerase activity and circumvents telomere-based replicative aging in normal cells. We have used hTERT to immortalize a variety of human cell types. Corneal epithelial cells expressing introduced hTERT require growth on collagen IV while both breast and skin epithelial cells require feeder layers to prevent a premature p16-induced growth arrest due to inadequate culture conditions. Human corneal and skin cells expressing hTERT can be used to form organotypic (3D) cultures that may be useful for regenerative medicine. Such organotypic cultures express differentiation-specific proteins, suggesting that hTERT does not alter the differentiation phenotype.

To immortalize human bronchial epithelial cells (HBEC) we introduced hTERT and Cdk4 (to bypass upregulated p16). In organotypic (3D) culture, immortalized bronchial cells are placed on top of collagen plugs contracted by lung fibroblasts and then exposed to an air-medium interface and allowed to differentiate. Under these conditions HBECs are able to differentiate into both mucous (goblet) and ciliated epithelial cells. If the same cells are placed on matrigel, the cells produce alveolar cysts and express surfactant proteins. This suggests that the immortalized HBECs have both bronchiolar and alveolar stem cell characteristics. These immortalized HBECs also provide a novel resource to study the molecular pathogenesis of lung cancer. In these immortalized HBEC cells we have knocked down TP53, and overexpressed c-Myc and the transforming oncogene K-rasv12. When introduced into immunosuppressed mice, these cells make both squamous cell and adenocarcinomas, suggesting immortalized HBECs have bronchiolar-alveolar stem-like characteristics.

Organotypic cultures bridge standard monolayer cultures (2D) with those of animal models of lung cancer, permitting a fuller understanding of the pathogenesis of lung cancer. In summary, the production of hTERT engineered cells offer the possibility to model a variety of diseases and aged-related medical conditions that are due to telomere-based replicative senescence.

Keywords: Cancer, Tissue Engineering, Telomeres, Aging