UNILINEAGE MEGAKARYOCYTIC PROLIFERATION AND DIFFERENTIATION OF PURIFIED HEMATOPOIETIC PROGENITORS IN SERUM-FREE LIQUID CULTURE

Cellular and molecular analysis of megakaryocytopoiesis has been hampe red thus far by the lack of pure and abundant megakaryocyte (MK) cell populations, In this study, hematopoietic progenitor cells (HPCs), str ingently purified from peripheral blood, were induced to megakaryocyti c differentiation/maturation in serum-free liquid suspension culture t reated with a growth factor cocktail (interleukin-3 [IL-3], c-kit liga nd, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal matur ation; (2) further addition of mpIL increased the MK purity level to 8 0% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell n umber (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphologi cal evaluation indicated the presence of putative mononuclear MK precu rsors and then of mature polynucleated platelet-forming MKs, peaking a t days 5 and 12, respectively, Membrane phenotype analysis showed a gr adual decrease of CD34(+) HPCs, coupled with an inverse increase of MK -specific antigens (eg, CD61/62/42b) starting before mature MK detecti on by morphology analysis, In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature M Ks. Furthermore, MKs synthesize and secrete low but significant amount s of both IL-6 and granulocyte-macrophage colony-stimulating factor, C omparative culture studies were performed on purified bone marrow CD34 (+)/38(hi) or CD34(+)/38(lo) cells stimulated by mpIL alone. Both popu lations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no prolifera tion, In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of high ly purified or ''pure'' megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis. (C) 1995 by The American Society of Hematology.