Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34+ cells

12. Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34+ cells Pages 460-470 Stefan Scheding, Bernhard Meister, Hans-Jörg Bühring, Charles M. Baum, John P. Mc Kearn, Thomas Bock, Lothar Kanz, Wolfram Brugger Preview Purchase PDF (476 K) | Related Articles Abstract | Figures/Tables | References Abstract Objective Neutropenia following high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation might be abrogated by an additional transplantation of ex vivo generated granulopoietic postprogenitor cells (GPPC). Therefore, the ex vivo expansion of CD34+ PBPC was systematically studied aiming for optimum GPPC production. Materials and Methods CD34+ PBPC were cultured in serum-free medium comparing different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), granulocyte colony-stimulating factor (G), granulate-macrophage colony-stimulating factor (GM), daniplestim (D, a novel IL-3 receptor agonist), and Flt3 ligand (FL) under various culture conditions. Ex vivo generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. Results Addition of G ± GM but not GM alone to cultures stimulated with S163E effectively induced the generation of GPPC. GPPC production was maximum after 12 to 14 days. Best expansion rates were observed when cells were cultured at 1.5 × 104/mL in 21% O2. Modifications of culture conditions were either less or equally effective (i.e., modification of starting cell concentrations, low oxygen, addition of serum albumin or autologous plasma, repetitive feeding). Comparison of different cytokine combinations revealed that the optimum GPPC expansion cocktail consisted of S6GD+FL (day 12: 130-fold cellular expansion, 32% myeloblasts/promyelocytes, 49.4% myelocytes/metamyelocytes, 12.4% bands/segmented), which furthermore expanded CD34+ cells (3.4-fold) and clonogenic progenitors (13.4-fold). Conclusion Using the S6DG+FL expansion cocktail, GPPC could be effectively produced ex vivo starting from positively selected CD34 PBPC, possibly enabling amelioration or even abrogation of posttransplant neutropenia.