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Ex Vivo Generation of Megakaryocyte Precursors from Umbilical Cord Blood CD34+ Cells in a Closed Preclinical System



Ex Vivo Generation of Megakaryocyte Precursors from Umbilical Cord Blood CD34+ Cells in a Closed Preclinical System



Blood 100(11): Abstract No. 5247



Umbilical cord blood (UCB) provides a rich source of stem cells for hematopoietic reconstitution after myeloablative therapy. A major disadvantage of UCB is delayed engraftment of platelets, which can lead to hemorrhagic complications, increased platelet transfusion requirements, prolonged hospitalization and allo-immunization. We propose to accelerate platelet engraftment by expanding the number of megakaryocyte (MK) precursors through cytokine stimulation. Cord blood was Ficoll-separated and frozen for subsequent use. Upon thawing, the mononuclear cell (MNC) population was positively selected for CD34 expression with MACS cell sorting (Miltenyi Biotec). The cells were cultured in closed, gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: r-huIL-3 (100ng/ml) + r-huFlt-3 + r-huSCF + r-huTPO (all 50ng/ml). MK precursor expansion was assessed by MNC count and flow cytometry (CD41, CD34/41, CD61 and CD34/61 expression) on days 7, 11 and 14. Stem cell expansion was quantitated via staining for CD34. Twelve experiments were performed under these identical conditions. The optimal expansion of CD41 and CD34/41 cells was observed at day 11, with a median of 25 (range 5-83) CD41 cells and 4 (range 1-42) CD34/41 cells generated per CD34 cell seeded at day 0. At day 11 there were 6 (range 3-54) CD61 and 3 (range 1-31) CD34/61 cells generated per CD34 cell at day 0. Expansion of MNCs at day 11 was 54-fold (range 6-152) and CD34 cells 6-fold (range 2-54) respectively. Engraftment data suggests that the generation of 4 CD34/41 cells per CD34 cell cultured should significantly hasten platelet engraftment in UCB recipients. Previous ex vivo stem cell expansion protocols have been developed to increase MNC and CD34 cell number to facilitate neutrophil engraftment. However, none have focused on clinical expansion of MK precursors to facilitate platelet recovery. Our next goal will be to determine if MK precursor expansion in an aliquot of stem cells leads to earlier platelet engraftment without compromising other short- and long-term engraftment kinetics.

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