Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGF beta.

Abstract

The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml, and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells, the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes, without interference from factors in the serum.