LONG-TERM MAINTENANCE OF HEMATOPOIESIS IN IRRADIATED MICE BY RETROVIRALLY TRANSDUCED PERIPHERAL-BLOOD STEM-CELLS

Mobilized peripheral blood stem cells (PBSC) are used as a source of h ematopoietic stem cells for transplantation and gene therapy, It is st ill unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to prov ide long-term function of transgene in reconstituted mice, In the pres ent study, mobilized PBSC from male mice were transduced with human ad enosine desaminase gene (hADA) and were used for reconstitution of let hally irradiated female mice. At 1 1/2, 3, 6, 9, and 12 months after r econstitution, the bone marrow cells were repeatedly collected from ea ch 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 CFUS-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-chrom osome-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 m obilized PBSC or bone marrow cells. However, in both cases the CFU-S c ontent in the bone marrow was reduced fivefold to 10-fold compared wit h the concentration of CFU-S in mice transplanted with nontransduced b one marrow. The SRA of CFU-S in mice reconstituted with peripheral blo od 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 P BSC. The integration of the human ADA gene was observed during 9 month s in about 60% of studied CFU-S. The proportion of marked colonies sha rply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridiz ation in the same reconstituted mice during the whole period of observ ation, The time of clone existence was about 3 months. We conclude tha t long-term marrow repopulating cells mobilized into circulation by tr eatment with granulocyte colony-stimulating factor (G-CSF) and stem ce ll factor (SCF) are capable of maintaining lifelong polyclonal hematop oiesis in reconstituted mice. (C) 1997 by The American Society of Hema tology.