HUMAN UMBILICAL CORD BLOOD CD34+ CELL EXPANSION IN THE PRESENCE OF FLT-LIGAND(FL). 133

Human umbilical cord blood (HUCB) is an important source of progenitor cells which are able to reconstitute hematopoiesis. The in vitro expansion of isolated CD34 positive cells should provide a sufficient number of hematopoietic cells for transplants in large reciplents and would increase vector-mediated gene transfer efficiency. In vitro expansion of progenitor cells requires the identification of the cytokine combinations which result in the greatest increase of committed cells, without exhausting the early progenitor pool. In this study we tested the effect of three colony stimulating-factor (CSF) combinations [SCF^*+IL-6(CSF2); SCF^*+IL-6+FL (CSF3); SCF^*+IL-6+FL+IL-3 (CSF4)] on CD34+ cells isolated(MiniMACS, Miltenyi Biotec, Germany) from HUCB. CD34+ cells, ≥97% pure in all samples, were ≥97% CD33- (range 97-99%) and ≥97% CD38+ (range 97-99.8%). The absolute number of total cells and cell subsets, evaluated after 7 and 11 days of liquid cultures, showed the following median fold increase:Table

Table 1

CD34+ cells were induced to proliferate mostly in the presence of FL. Freshly isolated CD34+ cells, as well as CD34+ cells expanded in liquid cultures, were tested for growth of CFU-GM. Absolute numbers of these colonies, scored after 7 and 14 days in semisolid medium culture, showed an overall increase of the colony forming cells, with an additive effect when FL had been included in the liquid culture expansion. These data confirm that ex vivo expansion is feasible for CD34+ isolated cells. The ex vivo expansion conditions adopted demonstrated that a higher CD34+ amplification was obtained in the presence of FL. Further addition of IL-3 strongly affected CD34+CD38- cell subset expansion. Extensive proliferation in presence of CSF2, combined with FL alone or FL+IL-3, did not exhaust the early progenitor cell pool.