Thrombopoietin Requires Additional Megakaryocyte-Active Cytokines for Optimal Ex Vivo Expansion of Megakaryocyte Precursor Cells

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Abstract
Little is known concerning the interaction of thrombopoietin (TPO) with other megakaryocyte-active cytokines in directing the early events of megakaryocyte development. Culture of CD34+ cells in interleukins (IL) -1, -6, -11, plus stem cell factor (SCF; S) results in a 10- to 12-fold expansion in total cell numbers, whereas total CD41+ megakaryocytes are expanded ∼120-fold over input levels. Addition of TPO to IL-1, -6, -11, S generates a biphasic proliferation of CD41+ cells, accelerates their rate of production, and results in an ex vivo expansion of more than 200-fold. The addition of Flt-3 ligand (FL) increases CD41+ cell expansion to ∼380-fold over input levels. In the absence of TPO, ∼95% of the expanded cells show the phenotype of promegakaryoblasts; TPO and/or FL addition increases CD41 antigen density and ploidy in a subpopulation of promegakaryoblasts. A moderate (approximately sevenfold) expansion of megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an ∼17-fold expansion. We conclude that early proliferative events in megakaryocyte development in vitro are regulated by multiple cytokines, and that TPO markedly affects these early developmental steps. However, by itself, TPO is neither necessary nor sufficient to generate a full proliferative/maturational in vitro response within the megakaryocyte compartment. TPO clearly affects terminal differentiation and the development of (some) high-ploidy human megakaryocytes. However, its limited in vitro actions on human cell polyploidization suggest that additional megakaryocyte-active cytokines or other signals are essential for the maximal development of human megakaryocytes.