Mori Pg

EFFICIENCY OF RETROVIRAL TRANSDUCTION OF CD 34+ CELLS FROM UMBILICAL CORD BLOOD DEPENDS ON GROWTH FACTOR STIMULATION. 134

The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of stable transduction. The great potentiality of umbilical cord blood CD34+ cells prompted us to evaluate which factor(s) play a major role(s) in influencing the transduction efficiency of retroviral vectors. CD34+ cells, isolated with an immunomagnetic device (MiniMacs, Miltenyi Biotech, Germany), 90-95% pure as assessed by cytofluorimetric analysis, were transduced with the LXSN retroviral vector carrying the neomicine resistance gene. Infection was made with virus-containing supernatant of Am12 env+ packaging cell line, having a titer of 2×106 PFU/ml. All the infections were performed using a multiplicity of infection of 10 PFU/cell. Multiple or single infections were performed with the following schedules: i) up to 6 infection lasting one hour over 3 days; ii) a 72 hr infections substituting fresh viral supernatant and cytokines every 24 hrs; iii) a single 72 hr infection. In most experiments, CD34+ cells were preincubated with different cytokine cocktails for 24 or 48 hrs before infection. Efficiency of transduction was evaluated both by clonogenic assays and semiquantitative PCR analysis performed either immediately or after 7 day expansion in the presence of cytokines and G-418. G-418-resistant CFU-E, BFU-E and CFU-GM colonies were scored after 7 and 14 days. We provided evidence that the efficiency of transduction was mainly dependent on cell preincubation with growth factors rather than on the number of infections. Precisely, infection performed on freshly isolated CD34+ was almost ineffective (0.4%) while 48 hr preincubation in the presence of SCF*+IL-6+IL-3 allowed to reach a level of transduction up to 40% regardeless of the number of infections. In this respect it is noteworthy that a single infection, lasting as long as a multiple infection course gave identical results. In conclusion the results obtained suggest that high efficiency of transduction of umbilical cord blood CD34+ cells can be achieved in liquid, stroma free, cultures.

PSYCHOLOGICAL AND SOCIAL COMMITMENT IN GENETIC COUNSELLING AND IN PRENATAL DIAGNOSIS OF HEMOPHILIA 190

PSYCHOLOGICAL AND SOCIAL COMMITMENT IN GENETIC COUNSELLING AND IN PRENATAL DIAGNOSIS OF HEMOPHILIA 190

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