Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34⁺ cells in collagen and liquid culture media

Umbilical cord blood (UCB) is now commonly used as a source of stem cells for hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, platelet engraftment occurs at a median of 71 days which is significantly prolonged compared to allogeneic bone marrow. The number of megakaryocyte (MK) precursors in stem cell harvests appears to correlate inversely with the time to platelet engraftment. In an effort to increase the number of platelet precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41⁺) and stem cells (CD34⁺ and AC133⁺ cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p < 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34⁺, AC133⁺, CD41⁺ and CD61⁺ cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.