EX-VIVO EXPANSION OF MEGAKARYOCYTE PROGENITORS - EFFECT OF VARIOUS GROWTH-FACTOR COMBINATIONS ON CD34(-MARROW AND G-CSF-MOBILIZED PERIPHERAL-BLOOD() PROGENITOR CELLS FROM BONE)

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK ) progenitor cell reconstitution is a serious complication of hematopo ietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clin ically beneficial. In this study we investigated the ability of variou s growth factor combinations to generate MK progenitors. CD34(+) cells derived from bone marrow (BM) and granulocyte colony-stimulating fact or (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breas t 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: St em cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1) ; SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (com bination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); a nd SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34(+) cells yie lded significantly higher numbers of CD41(+) MK progenitors than BM CD 34(+) cells with any of the growth factor regimens assayed. PB CD34(+) cells (2x10(5)) at day 0 generated 1.2 to 1.3x10(6) CD41(+) cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34(+) cells produced 5x10(5) CD41() cells after 9 days in the presence of combination 1, whereas lower n umbers of CD41(+) cells were generated in cultures with combinations 2 and 3 (2.3x10(5) and 4.2x10(4), respectively). The addition of MGDF t o cultures that were grown with combination 1 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 res ulted in higher frequencies of MK progenitors than those obtained in c ultures 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 (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemical ly identified megakaryocytic cells revealed mononuclear cells as the p redominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, ev en under the influence of MGDF, whereas in BM-derived cultures MGDF in duced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majorit y of cultured megakaryocytic cells coexpressed CD34 and platelet glyco proteins (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 hig h interpatient variations. There was no correlation between platelet r ecovery following HDC with hematopoietic cell support and the magnitud e of GPI cell expansion ex vivo, suggesting the feasibilty of MK expan sion ex vivo in patients with prolonged thrombocytopenia posttransplan tation. In summary, these data indicate that GCSF-mobilized CD34(+) PB PCs are more effectively expanded ex vivo into the megakaryocytic line age than are CD34(+) BMPCs. CD34(+):GP(+) MK progenitors may be an app ropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.