Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium

Megakaryocytes (MK) were expanded from purified human CD34⁺ cells obtained from three sources, bone marrow (BM), mobilized peripheral blood progenitor cells (PB), and umbilical cord (UC) blood. CD34⁺-selected cells were cultured for 12 days with 10 ng/ml thrombopoietin (TPO), 10 ng/ml IL-3, 10 ng/ml TPO + 10 ng/ml IL-3, or 200 ng/ml promegapoietin (PMP), a chimeric dual agonist of the c-MpI and human IL-3 receptors. MK production was compared in serum-free versus human serum-supplemented liquid media. PMP and the combination of TPO and IL-3 (TPO + IL-3) increased MK production similarly. Culturing CD34⁺ cells with PMP in serum-free medium resulted in a twofold increase in MK yield compared with serum-supplemented medium. CD34⁺ cells from UC proliferated more than those from either BM or PB in liquid culture, resulting in much greater MK production under all conditions. Phenotypic analysis of the uncultured CD34⁺ cells showed that BM had a higher frequency of CD34⁺/CD41⁺ cells than PB or UC. TPO + IL-3 or PMP produced larger and greater numbers of BFU-MK and CFU-MK per seeded CD34⁺/CD41⁺ cell from UC than from either BM or PB. Thus, although uncultured CD34⁺-selected BM cells contained a higher frequency of committed mature MK progenitors, UC CD34⁺ cells had a greater proliferative capacity and, therefore, were more productive. PMP induced megakaryocytopoietic activity comparable to that achieved with TPO + IL-3 and may be useful for ex vivo expansion of MK for clinical trials.