Ex vivo expansion of megakaryocyte (MK) progenitors

Purpose: Prolonged thrombocytopenia due to inadequate MK progenitor cell reconstitution is a serious complication, and can be the cause of death in patients undergoing high-dose chemotherapy and hematopoietic cell support. 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 5 growth factor combinations (GFC) to induce proliferation of MK progenitors. Methods: Human CD34-positive (+) cells from bone marrow (BM, n=8) and G-CSF mobilized peripheral blood (PB, n=9) were cultured unpertubed for 10 days at 2×10⁴ cells/ml with Defined Media (Amgen Inc.); at day 0, the following GF were added to the media: SCF, IL-3, IL-6, G-CSF, and MGDF. Cultures were sampled daily by immunocytochemistry and flow cytometry, using anti-CD61, anti-CD41, and anti-CD42a monoclonal antibodies. Results: PB CD34⁺ cells yielded significantly higher numbers of MK progenitors than BM CD34⁺ cells with any of the GFC used. In particular, 2×10⁴ CD34⁺ cells at day 0 generated 1.2-1.3×10⁵CD41⁺ cells by day 10, when cultured with SCF, IL-3, IL-6+G-CSF (GFC1), SCF, MGDF+G-CSF (GFC2), or SCF+MGDF (GFC3). In contrast, 2×10⁴ BM CD34⁺ cells produced of 5×10⁵ CD41⁺ cells by day 9 in the presence of GFC1, whereas lower numbers of CD41⁺ cells were obtained in cultures with GFC2 and GFC3. However, addition of MGDF to cultures which were grown with GFC1 for 5 days, further increased the number of MK progenitors in BM-derived cultures as well as in PB-derived cultures. Morphological analysis of MK progenitors revealed a mononuclear cell as the predominant cell type during the 10 day culture period. No significant maturation was observed in PB-derived cultures, whereas in BM-derived cultures MGDF induced a significant shift to more mature MK. Additionally, flow cytometric analysis showed that 100% of MK in PB-derived cultures coexpressed CD34 and MK antigens until day 8 of culture, also indicating an early stage of development. Conclusion: PB CD34⁺ cells are a more suitable source for ex vivo expansion of MK progenitors than BM CD34⁺ cells. The reconstitutive potential of the ex vivo expanded cells will be studied in a prospective clinical trial.