Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture

Abstract

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.

A combination of megakaryocyte growth and development factor and interleukin‐1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes

“…In contrast, no proliferation but only megakaryocyte differentiation was observed in cultures in the presence of PEG-rHuMGDF alone (data not shown). Thus, as described by others, PEGrHuMGDF must be combined with other growth factors to achieve the proliferation of megakaryocytes (Gehling et al, 1997;Guerriero et al, 1995;Choi et al, 1996;Birkmann et al, 1997;Borge, et al, 1997;Dolzhanskiy et al, 1997;Debili et al, 1995). In our culture system, IL-1 was found to induce the highest proliferation in combination with PEG-rHuMGDF.…”

“…TPO has been shown to be the major regulator in the process of differentiation, proliferation and maturation of stem cells into megakaryocytes. Moreover, TPO appears to be essential for optimal megakaryocyte expansion ex vivo (Gehling et al, 1997;Guerriero et al, 1995;Debili et al, 1995;Nichol et al, 1995;Wendling et al, 1994). To date, clinical trials have shown that administration of recombinant TPO does not result in a satisfactory reduction in the duration of chemotherapyinduced thrombocytopenia Nash et al, 1997;Bolwell et al, 1997;Linker et al, 1998).…”

“…A broad range of cytokines, i.e. TPO (pegylated megakaryocyte growth and development factor (PEGrHuMGDF), a truncated, pegylated mpl ligand related to TPO), stem cell factor (SCF), interleukin 1 (IL-1), IL-3, IL-6, IL-11, erythropoietin, granulocyte macrophage-colony stimulating factor (GM-CSF),¯t3-ligand, macrophage in¯ammatory protein-1a and G-CSF, have been tested, and different cytokine cocktails have been shown to be able to stimulate megakaryocytopoiesis in vitro (Gehling et al, 1997;Guerriero et al, 1995;Birkmann et al, 1997;Dolzhanskiy et al, 1997;Debili et al, 1995;Bertolini et al, 1997;Catani et al, 1998;Ohmizono et al, 1997;Williams et al, 1998). However, inclusion of large numbers of cytokines in an ex vivo expansion protocol would be too expensive for routine clinical use.…”

“…IL-1 also has been found to enhance megakaryocyte proliferation Williams et al, 1998). IL-3 increases the absolute number of megakaryocytes in liquid culture (Guerriero et al, 1995;Debili et al, 1995Debili et al, , 1991 and expands the number of megakaryocyte colonies (Dolzhanskiy et al, 1997(Dolzhanskiy et al, , 1998Catani et al, 1998). SCF has been shown to promote colony formation of haemopoietic stem cells in combination with other growth factors (IL-3, IL-6, G-CSF, GM-CSF) (Migliaccio et al, 1992;Briddell et al, 1991).…”

Effects of Recombinant Human Thrombopoietin Alone and in Combination with Erythropoietin and Early‐Acting Cytokines on Human Mobilized Purified CD34 + Progenitor Cells Cultured in Serum‐Depleted Medium

“…In the absence of EPO and early-acting cytokines like IL-3, SCF and GM-CSF, TPO at first sight acts as a strictly lineage-specific cytokine: most CD34 + cells are driven into the megakaryocyte-platelet lineage within 12 days of culture. Guerriero et al [41] recently reported a unilineage megakaryocytic proliferation and differentiation in a similar assay system. Yet the effects of TPO alone were crucially dependent on the culture period; the molecule acted on different target cell populations during the first and second week of culture.…”