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Unilineage megakaryocytic proliferation and differentiation of purified
hematopoietic progenitors in serum-free liquid culture
R Guerriero, U Testa, M Gabbianelli, G Mattia, E Montesoro, G Macioce, A Pace, B Ziegler, HJ Hassan and C Peschle
Department of Hematology-Oncology, Istituto Superiore di Sanita, Rome,
Italy.
Cellular and molecular analysis of megakaryocytopoiesis has been hampered
thus far by the lack of pure and abundant megakaryocyte (MK) cell
populations. In this study, hematopoietic progenitor cells (HPCs),
stringently purified from peripheral blood, were induced to megakaryocytic
differentiation/maturation in serum-free liquid suspension culture treated
with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, 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 maturation; (2) further
addition of mpIL increased the MK purity level to 80% 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 number (to 1.5 x 10(5) cells). In
mpIL-supplemented culture, morphological evaluation indicated the presence
of putative mononuclear MK precursors and then of mature polynucleated
platelet- forming MKs, peaking at days 5 and 12, respectively. Membrane
phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an
inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before
mature MK detection by morphology analysis. In situ hybridization showed
the expression of MK-specific von Willebrand gene in both MK precursors and
mature MKs. Furthermore, MKs synthesize and secrete low but significant
amounts of both IL-6 and granulocyte- macrophage colony-stimulating factor.
Comparative culture studies were performed on purified bone marrow
CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations
generated a highly enriched MK progeny (62% and 93% MKs at day 12 of
culture, respectively) but showed either little or no proliferation. In
conclusion, the purified peripheral blood HPC differentiation culture
system allows for growth of a relatively large number of highly 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.
Volume 86,
Issue 10,
pp. 3725-3736,
11/15/1995
Copyright © 1995 by The American Society of Hematology
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