STROMAL CELLS MAINTAIN THE RADIOPROTECTIVE CAPACITY OF CFU-S DURING RETROVIRAL INFECTION

Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection pro tocols which preserve the radioprotective capacity of CFU-S, we design ed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived colonies from bone marrow cells of 5-FU-treated mice wer e tested for their capacity to prevent radiation-induced mortality. Th ree parameters which may modify stem cell potential were tested in inf ection protocols using a retroviral vector containing the gene for neo mycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell l ine by a 'competent' stromal cell line; and (3) the effects of G418 se lection. All CFU-S having radioprotective capacity were found in the a dherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow cult ure were used-during the co-culture. The neo resistance gene was trans duced into CFU-S with the same efficiency using co-culture with the pa ckaging cell line or co-culture with the MS-5 cell line plus viral sup ernatant. However, in the presence of MS-5, a much higher proportion o f CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoie tic reconstituting ability. Finally, G418 selection, even for a limite d period (24 h), significantly decreased the radioprotective capacitie s of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitutio n 1 by single CFU-S was quantified in recipient mice. The progeny of C FU-S were found at a significant level in the blood, spleen and bone m arrow in 38% and 15% of mice, 1 and 3 months after transplantation res pectively. These results demonstrate that we have substantially improv ed the infection protocol. Under these conditions of infection, if is possible to conserve CFU-S properties and to transduce a gene into a s tem cell with short-term hematopoietic reconstitution potential.