Phenotypical and functional characterizations of ex vivo manipulated cd34± progenitors from steady state and mobilized peripheral blood

Cytokine mobilized peripheral blood progenitor cells are widely used lor iranspl.ini.iiu'n in cancer patients because of easy collection and rapid hématologie reconstitution These cells may be appropriate targets for gene transfer. Therefore, We investigated the effects of ex vivo manipulation on hematopoietic reconstitution potential of these cells. Immunoselected CD34± progenitors isolated from steady-state (ssCD34±) or G-CSF mobilized (mCD34±) peripheral blood were cultured for 5 days: 2-day liquid culture followed by 3-day coculture on A7.21, a rnurine fibroblast layer, in the presence of 10% PCS, 1L-3, IL-6, GM-CSF, and KL. Higher levels of expansion in CD34± cells were observed in ssCD34± than in mCD34± cells (4.6±3.3- vs. 2.2±1.3-fold). Hematopoietic reconstitution potentials of these ex vivo manipulated cells were evaluated using the SCID-hu mouse models implanted with human fetal bone marrow (BM) or thymus/liver. Human BM analyzed 4 weeks after injection of manipulated ssCD34± cells showed reconstitution in CD19± (0.6-98.8%), CD33± (2.8-99.3%) and CD34± progenitor (2.4-17%) cells (n=4); ihyinic grafts analyzed 6-8 weeks after injection also demonstrated high levels of T cells reconstitution (67.2-86.4%) (n=4). In contrast, only very low levels of reconstitution (