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 f or hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, p latelet engraftment occurs at a median of 71 days which is significantly pr olonged 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 platel et precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cel ls 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 er ythropoietin (Epo); or all six cytokines in combination. After 16 days, sig nificant expansion of MK precursors (CD41(+)) and stem cells (CD34(+) and A C133(+) cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 wit h or without GM-CSF compared to the combinations that contained Epo (p< 0.0 5). Similar studies were performed using liquid culture medium, and after 1 4 days the number of MNCs, CD34(+), AC133(+), CD41(+), and CD61(+) cells we re higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared t o those cultured with those four cytokines plus GM-CSF. These results demon strate 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 addi tion of Epo and GM-CSF is unnecessary.