Ex vivo expansion of megakaryocyte progenitors: Effect of various growth factor combinations on CD34⁺ progenitor cells from bone marrow and G-CSF- mobilized peripheral blood

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34' cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL- 6 + G-CSF (combination 1); 5CF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34⁺ cells yielded significantly higher numbers of CD41⁺ MK progenitors than BM CD34⁺ cells with any of the growth factor regimens assayed. PB CD34⁺ cells (2x10⁵) at day 0 generated 1.2 to 1.3x10⁶ CD41⁺ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10⁵ BM CD34⁺ cells produced 5x10⁵ CD41⁺ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41⁺ cells were generated in cultures with combinations 2 and 3 (2.3x10⁵ and 4.2x10⁴, respectively). The addition of MGDF to cultures that were grown with combination I for 5 days increased the number of CD41⁺ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB- derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10⁵ in PB- derived cultures, 7x10⁴ in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage 1 MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34⁺ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP⁺ cell expansion ex vivo, suggesting the feasibility of MK expansion ex vivo in patients with prolonged thrombocytopenia post-transplantation. In summary, these data indicate that G-CSF-mobilized CD34⁺ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34⁺ BMPCs. CD34⁺/GP⁺ MK progenitors may be an appropriate cell population for transplantation as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.