The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia

Abstract

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G‐CSF, GM‐CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi‐solid cultures to assess megakaryocyte colony growth (CFU‐Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU‐Mk growth from normal marrow increased 3–4‐fold and CD61+ve cells in suspension culture increased 8–10‐fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P < 0.05). IL6, GM‐CSF, G‐CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU‐Mk growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCF and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.

Megakaryopoiesis in vitro in myelodysplastic syndromor and development es and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors

“…Despite poor CFU‐Mk growth, the majority of patients and all normal samples showed a significant increase in CD61‐positive cells in suspension culture when stimulated with PEG‐rHuMGDF, and in general only the addition of SCF or IL3 further enhanced the response. These effects were consistent with our previous findings in samples from aplastic anaemia patients ( Brereton et al , 1999 ). Another study has shown that PEG‐rHuMGDF stimulation of single human megakaryopoietic progenitor cells to divide can be increased by the addition of SCF or IL3 ( Rasko et al , 1997 ).…”

In Vitro Proliferation and Differentiation of Megakaryocytic Progenitors in Patients with Aplastic Anemia, Paroxysmal Nocturnal Hemoglobinuria, and the Myelodysplastic Syndromes

“…In this study, we present data on Mk colony formation from purified BM CD34 + cells from patients with AA, hemolytic PNH, and MDS. Previous studies [10,17,20,22] have used unpurified BM cells, which makes analysis of the results more difficult, since contaminating accessory cells release a variety of hemopoietic growth factors and confound the interpretation of results. As with all other clonogenic assays there was considerable interindividual variation, even among the normal donors.…”

“…Crossover studies using CD34 + cells cultured onto normal stroma again show the absence of progenitor cell production, suggesting a deficiency of longterm culture initiating cells [6], which have been quantified in several studies and shown to be reduced [7][8][9]. To date, however, there has only been one published paper describing megakaryocytopoiesis in AA [10] from unpurified BM cells which showed significantly reduced megakaryocyte colonyforming units (CFU-Mks) compared with levels in normal donors. In patients with paroxysmal nocturnal hemoglobinuria (PNH), in vitro culture studies have shown a marked decrease in erythroid and myeloid progenitor cells in peripheral blood and BM [11][12][13][14][15][16], but there are very few data on megakaryocytopoiesis in PNH [12].…”

Recombinant human thrombopoietin (rhTPO) after autologous bone marrow transplantation: a phase I pharmacokinetic and pharmacodynamic study

“…20 Both the full length intact molecule (TPO 1-332) and peptide fragments derived from the amino terminus (TPO 1-153) are capable of differentiating progenitor cells into megakaryocytes and increasing the platelet count following in vivo administration. 21 TPO is the most effective cytokine to hasten recovery from thrombocytopenia in in vitro and in vivo models of severe thrombocytopenia. [22][23][24] Recombinant human thrombopoietin (rhTPO; Genentech, South San Francisco, CA, USA; Pharmacia and Upjohn, Bridgewater, CT, USA) has been entered into a broad array of clinical trials in the setting of myelosuppressive and myeloablative cytotoxic therapy.…”