Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.