In vitro growth and genetic manipulation of hemopoietic repopulating cells

The adult bone marrow contains stem cells capable of reconstituting all hemopoietic lineages for the life-time of a recipient following lethal irradiation and bone marrow transplantation. Despite more than two decades of research to characterize this population of pluripotent stem cells, mechanisms that regulate the survival, proliferation and differentiation in the initial stages of hemopoietic development in vivo remain poorly understood. Methods for maintaining, expanding and following the fate of the most primitive hemopoietic cells in vitro thus offers major opportunities for the investigation into these mechanisms. The major objectives of this work were therefore initially to determine if totipotent stem cells capable of repopulating hemopoiesis in lethally irradiated mice could be maintained and proliferate for extended times in vitro, and subsequently to quantitate stem cell numbers in these cultures and assess their in vivo repopulating potential in lethally irradiated hosts. Initial studies were aimed at determining if stem cells capable of repopulating all hemopoietic lineages in lethally irradiated mice could be maintained and proliferate when grown in a long-term culture (LTC) system. Marrow cells from 5-fluorouracil treated male mice were infected with a helper-free recombinant virus carrying the neomycin resistance gene and seeded onto irradiated adherent layers of pre-established, long-term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into multiple recipients. Southern blot analysis of hemopoietic tissues 45 days post transplant demonstrated large clonal populations common to lymphoid and myeloid tissues as indicated by the presence of unique retroviral insertion fragments. In a number of cases it was found that multiple recipients of a single flask were repopulated with the same clonally marked totipotent cells indicating expansion of such cells during culture prior to their injection into irradiated recipients. These results demonstrated for the first time both maintenance and expansion in vitro of totipotent stem cells with in vivo repopulating ability.Medicine, Faculty ofMedical Genetics, Department ofGraduat