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

Abstract

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-1β (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 induced 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-1β 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 40.7 ± 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 CD61 + , CD41 + , and CD42b + cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (vWF) (76.7 ± 3.3%, 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 be favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia.