Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice.

OBJECTIVE: We previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential. MATERIALS AND METHODS: Cord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice. RESULTS: In the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34(+)CD38-, p=0.001 and CD34(+)Lin-, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03). CONCLUSION: TEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34(+)CD38- and CD34(+)CD38- Lin- during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.

Microbial growth comparisons of five commercial parenteral lipid emulsions.

The ability of parenteral lipid emulsions to support microbial growth was compared using commercially available brands of lipid emulsion. Both 10 and 20% concentrations of soybean and safflower oil emulsions were used. Washed cultures of six gram-negative, three gram-positive, and one yeast, in concentrations of 1 x 10(4) to 2 x 10(4) colony-forming units/ml, were inoculated into lipid emulsion aliquots and stored at room temperature. There were than subcultured at 0, 6, 12, 24 and 48 hr. After 48 hr at 37 degrees C, growth was recorded as colony-forming units/ml. Normalized growth curves were expressed as mean +/- SEM. ANOVA demonstrated no difference in growth patterns due to the nature of the oil or its concentration. Gram-negative organisms multiplied faster when compared to gram-positive (p less than 0.05 at 12 hr, p less than 0.01 at 24 hr, and p less than 0.005 at 48 hr). Yeast grew as well as bacteria. The Center for Disease Control's recommendation of a 12-hr hang time for parenteral lipid emulsions should be observed until correlation of laboratory microbial growth patterns and clinical use are studied further.