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Evaluation of Cytokines for Expansion of the Megakaryocyte and Granulocyte Lineages

Northwestern University Department of Chemical Engineering, Evanston, Illinois, USA

Key Words. Megakaryocytes • Granulocytes • Progenitors • Serum-free medium • Liquid culture • CD34⁺ cells • Peripheral blood

Dr. William M. Miller, Northwestern University, Chemical Engineering Department, Evanston, IL 60208-3120, USA.

The goal of our study was to identify cytokine combinations that would result in simultaneous ex vivo expansion of both the megakaryocyte (Mk) and granulocyte lineages, since these cell types have the potential to reduce the periods of thrombocytopenia and neutropenia following chemotherapy. We investigated the effects of cytokine combinations on expansion of the Mk (CD41a⁺ cells and colony forming unit [CFU]-Mk) and granulocyte (CD15⁺ cells and CFU-granulocyte/monocyte [GM]) lineages. Peripheral blood CD34⁺ cells were cultured in serum-free medium with interleukin 3 (IL-3), stem cell factor (SCF), and various combinations of thrombopoietin (TPO), IL-6, GM-CSF, and/or G-CSF. The Mk lineage was primarily influenced by TPO in our cultures, although Mk and CFU-Mk numbers were increased when TPO was combined with IL-6. The primary stimulator of the granulocyte lineage was G-CSF, although many synergistic and additive effects were observed with addition of other factors. Expansion of CFU-GM increased upon addition of more cytokines. The cytokine combination of IL-3, SCF, TPO, IL-6, GM-CSF and G-CSF produced the greatest number of granulocytes and CFU-GM. The minimum cytokines necessary for expansion of both the Mk and granulocyte lineages included TPO and G-CSF, since no other factors examined could increase Mk and granulocyte numbers to the same extent. The number of hematopoietic progenitors produced in our culture system should be sufficient for successful engraftment following myelosuppressive therapy if produced on a scale of about one liter.

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