| Objectives To explore the optimal combination of cytokines and time of expansion umbilical cord blood (UCB) hematopoietic stem/progenitor cells (HSC/HPCs) in vitro to acquire sufficient HSC/HPCs for adult transplantation. Methods We have attempted to expand selected CD34+ cells in 2-week cultures in the presence various cytokine combinations including (1) stem cell factor (SCF), thrombopoietin (TPO), Flt3/Flk2 ligand (FL), (2) SCF, TPO, FL, IL-3, (3) SCF, TPO, FL, G-CSF in serum-free and stromal-free conditions. We have analyed total nucleated cells (TNC), colony-forming cell (CFC), telomerase activity and phenotyping of expanded cells after 7 d and 14 d. Results The highest folds amplification of TNC and CFC in the presence of IL-3 after 7d and 14 d of culture was obtained. The CD34+ cells with IL-3 was amplified in more than 10-fold as well as without IL-3 after 7 d of culture, and expansion fold of CD34+ cells was decreased slightly with IL-3 or G-CSF after 14 d culture. Proportion of CD33+ cells, CD41+ cells and CD71+ cells were increased after 7 d of culture in the conditions of three groups of cytokine combination and decreased 14 d of culture except CD41+ cells without IL-3. The telomerase activity was upregulated to the highest point companyed with stem/ progenitor cells extensively expansion after 7 d of culture including various cytokine combinations, but downregulated in the longer time culture. The OD titre of telomerase of expanded cells with IL-3 was lower than with G-CSF after 7d of culture (P=0.008). Conclusion Enough TNC and CD34+ cells to provide intermediate and rapid short-term engraftment for adult transplantation were acquired after 7 d expansion of a fraction of UCB CD34+ cells in vitro with cytokines support, which combined unmanipulated fraction of CB graft to provide stem cells for long-term engraftment. The optimal expansion time with IL-3 is 7 d or so, The ability of IL-3 to facilitate differentiation is higher than G-CSF in the presence of SCF, TPO and FL.
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