Effective ex vivo generation of megakaryocytic cells from mobilized peripheral blood CD34+ cells with stem cell factor and promegapoietin

Objective. The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced thrombocytopenia. Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for an optimum production of megakaryocytic cells. Materials and Methods. CD34(+) PBPC were cultured in serum-free medium comparing different (n = 23) combinations of stem cell factor (SCF) (S), IL-1 beta (1), IL-3 (3), IL-6 (6), erythropoietin (EPO) (E), thrombopoietin (TPO) (T) and promegapoietin (PMP, a novel chimeric IL-3/ TPO receptor agonist). Ex vivo-generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. Results. Addition of TPO to cultures stimulated with S163E, a potent progenitor cell expansion cocktail previously described by our group, effectively inducted the generation of CD61(+) cells (day 12: 31.4 +/- 7.9%). The addition of PMP tended to be more effective than TPO +/- IL-3. Whereas EPO was not required to maximize TPO- or PMP-induced megakaryocytic cell production, the use of IL-6 and IL-1 beta augmented cellular expansion as well as CD61(+) cell production rates in the majority of cytokine combinations studied. Thus, the most effective CD61(+) cell expansion cocktail consisted of S163 + PMP which resulted in 65.9 +/- 3.0% CD61(+) at day 12 and an overall production of 30.7 +/- 4.5 CD61(+) cells per seeded CD34(+) PBPC. However, the basic 2-factor combination S + PMP also allowed for an effective CD61+ cell production (day 12 CD61(+) cell production: 15.1 +/- 1.6), Moreover, maximum amplification of CFU-Meg was observed after 7 days using this two-factor cocktail (12.9 +/- 2.6-fold). The majority of CD61(+) cells generated in TPO- or PMP-based medium were low-ploidy 4N and 8N cells, and ex vivo-generated GD61(+), CD41(+), and CD42b(+) cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (VWF) (76.7 +/- 33%, 58.8 +/- 4.4%, and 82.7 +/- 2.5%, respectively). Conclusions. Taken together, this study demonstrates that megakaryocytic cells can be effectively produced ex vivo with as little as two-factors (SCF + PMP), an approach that might he favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.