Long-Term Maintenance of Hematopoiesis in Irradiated Mice by Retrovirally Transduced Peripheral Blood Stem Cells

Abstract

Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice. At 112, 3, 6, 9, and 12 months after reconstitution, the bone marrow cells were repeatedly collected from each mouse under light anesthesia and the number of colony-forming unit-spleen (CFU-S), spleen repopulating ability (SRA), and the integration of human ADA gene were studied in CFU-S–derived colonies by polymerase chain reaction (PCR) and Southern blot hybridization analyses. After 9 months, the proportion of donor CFU-S detected by PCR with a Y-chromosome–specific probe in mice reconstituted with mobilized PBSC was 75.3% ± 6.0%, which is similar to the concentration of donor CFU-S seen after bone marrow transplantation. Similarly, there was no difference in the concentration of CFU-S in mice reconstituted with transduced mobilized PBSC or bone marrow cells. However, in both cases the CFU-S content in the bone marrow was reduced fivefold to 10-fold compared with the concentration of CFU-S in mice transplanted with nontransduced bone marrow. The SRA of CFU-S in mice reconstituted with peripheral blood and bone marrow cells was the same 1.5 months posttransplantation, but after an additional 4 months, SRA of mice reconstituted with bone marrow cells was fivefold higher as compared with those engrafted by PBSC. The integration of the human ADA gene was observed during 9 months in about 60% of studied CFU-S. The proportion of marked colonies sharply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridization in the same reconstituted mice during the whole period of observation. The time of clone existence was about 3 months. We conclude that long-term marrow repopulating cells mobilized into circulation by treatment with granulocyte colony-stimulating factor (G-CSF ) and stem cell factor (SCF ) are capable of maintaining lifelong polyclonal hematopoiesis in reconstituted mice.