Reversible immortalization of human myogenic cells by site-specific excision of a retrovirally transferred oncogene

Myogenic cells have a limited life span in culture, which prevents expansio n at clinically relevant levels, and seriously limits any potential use in cell replacement or ex vivo gene therapy. We developed a strategy for rever sibly immortalizing human primary myogenic cells, based on retrovirus-media ted integration of a wildtype SV40 large-T antigen (Tag), excisable by mean s of the Cre-Lox recombination system. Myogenic cells were transduced with a vector (LTTN-LoxP) expressing the SV40 Tag under the control of an LTR mo dified by the insertion of a LoxP site in the U3 region. Clonal isolates of Tag-positive cells showed modified growth characteristics and a significan tly extended life span, while maintaining a full myogenic potential. Transi ent expression of Cre recombinase, delivered by transfection or adenoviral vector transduction, allowed excision of the entire provirus with up to >90 % efficiency. Cultures of Cre-treated (Tag(-)) or untreated (Tag(+)) myogen ic cells were genetically labeled with a lacZ retroviral vector, and inject ed into the regenerating muscle of SCID/bg immunodeficient mice. Tag(-) cel ls underwent terminal differentiation in vivo, giving rise to clusters of b eta-Gal(+) hybrid fibers with an efficiency comparable to that of control u ntransduced cells. Tag(+) cells could not be detected after injection. Neit her Tag(+) nor Tag(-) cells formed tumor in this xenotransplantation model. Reversible immortalization by Tag therefore allows the expansion of primar y myogenic cells in culture without compromising their ability to different iate in vivo, and could represent a safe method by which to increase the av ailability of these cells for clinical application.