The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors

Abstract

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin- 6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin‐induced differentiation

“…It could be argued that these differences could be a consequence of the experimental conditions, as only TPO was added to the culture medium. However, TPO alone is able to support in vitro maturation of megakaryocytes (Debili et al, 1995;Dolzhanskiy et al, 1997). Additionally, similar differences in ploidy levels have been observed in megakaryocytes cultured in various culture conditions from a range of fetal and adult sources (Vitrat et al, 1998b;Ma et al, 2000;Miyazaki et al, 2000).…”

Dose–response relationship of megakaryocyte progenitors from the bone marrow of thrombocytopenic and non‐thrombocytopenic neonates to recombinant thrombopoietin

“…This observation is concordant with previously published studies using cord blood progenitors (Schipper et al, 1998), but it differs from the results obtained by Nishihira et al (1996), who found that the optimal rTpo concentration was 50 ng/ml, and Murray et al (1998), who showed increasing numbers of colonies with rTpo concentrations as high as 100 ng/ml. Similarly, studies using progenitor cells from adults have described maximal colony growth using concentrations of rTpo as varied as 1 ng/ml (Teramura et al, 1997), 5 ng/ml (Debili et al, 1995) and 100 ng/ml (Angchaisuksiri et al, 1996). These differences are probably related to the different culture systems used, the presence of serum or serumderived products in the cultures (which might contain factors that affect MK colony growth) or the type of cells plated (unfractionated marrow cells, peripheral blood progenitors, CD34 1 cells, etc.).…”

The role of thrombopoietin in post‐operative thrombocytosis

“…Thrombopoietin (Tpo) is the main regulator of platelet production (Kaushansky, 1995) and, therefore, is a probable candidate to play a role in the platelet rise. Tpo alone can stimulate both proliferation and differentiation of megakaryocytic cells, which eventually results in the release of platelets in the circulation (Wendling et al, 1994;Choi et al, 1995;Debili et al, 1995a;Kaushansky et al, 1995;Cramer et al, 1997;Gehling et al, 1997;Norol et al, 1998). A potential increase in circulating Tpo after major surgery may arise via different mechanisms.…”