[Tissue donation in nursing homes; the situation two years after the introduction of the Organ Donation Act]. / Weefseldonatie in verpleeghuizen; de situatie 2 jaar na invoering van de Wet op de Orgaandonatie.

OBJECTIVE: To ascertain the state of affairs in Dutch nursing homes with regard to the Organ Donation Act, two years after its introduction. DESIGN: Descriptive, questionnaire study. METHOD: By means of a structured questionnaire the availability, implementation and use of the protocol on tissue donation as well as difficulties in the use of this protocol were enquired about in April 2000 in all 253 Dutch combined and categorical somatic nursing homes. The attitude of nursing home physicians to tissue donation in nursing homes was also assessed. The questionnaire was sent to heads of the Medical Departments, who supplied the answers in cooperation with the nursing home physicians. Finally, partly based on registration data from the Donor Register and from Bio Implant Services, an inventory was made of the number of occasions the national Donor Register was consulted and of the number of tissue donations from nursing homes. RESULTS: In total, 186 questionnaires were returned (response: 73.5%). A protocol on tissue donation had been introduced by 136 (73%) of the Dutch nursing homes, and 96 (52%) of the nursing homes actually used such a protocol. Since the introduction of the Organ Donation Act, the number of tissue donations from nursing homes has not increased: 1-2 skin and 15-20 cornea donors per year. The nursing home physicians were of the opinion that compliance with the protocol required a considerable amount of time, work and emotions, yet due to the large number of contraindications for donation, these efforts were scarcely rewarded.

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 expansion at clinically relevant levels, and seriously limits any potential use in cell replacement or ex vivo gene therapy. We developed a strategy for reversibly immortalizing human primary myogenic cells, based on retrovirus-mediated integration of a wild-type SV40 large-T antigen (Tag), excisable by means 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 modified by the insertion of a LoxP site in the U3 region. Clonal isolates of Tag-positive cells showed modified growth characteristics and a significantly extended life span, while maintaining a full myogenic potential. Transient 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+) myogenic cells were genetically labeled with a lacZ retroviral vector, and injected into the regenerating muscle of SCID/bg immunodeficient mice. Tag- cells underwent terminal differentiation in vivo, giving rise to clusters of beta-Gal+ hybrid fibers with an efficiency comparable to that of control untransduced cells. Tag+ cells could not be detected after injection. Neither Tag+ nor Tag- cells formed tumor in this xenotransplantation model. Reversible immortalization by Tag therefore allows the expansion of primary myogenic cells in culture without compromising their ability to differentiate in vivo, and could represent a safe method by which to increase the availability of these cells for clinical application.