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

Objective. Neutropenia following high-dose chemotherapy and peripheral bloo d progenitor cell (PBPC) transplantation might be abrogated by an additiona l 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 comp aring different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), gra nulocyte colony-stimulating factor (G), granulate-macrophage colony-stimula ting 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 maxim um after 12 to 14 days. Best expansion rates were observed when tells were cultured at 1.5 x 10(4)/mL in 21% O-2. Modifications of culture conditions Here 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 reveale d that the optimum GPPC expansion cocktail consisted of S6GD + FL (day 12: 130-fold cellular expansion, 32% myeloblasts/promyelocytes, 49.4% myelocyte s/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 effective ly produced es vivo starting from positively selected CD34 PBPC, possibly e nabling amelioration or even abrogation of posttransplant neutropenia, (C) 2000 International Society for Experimental Hematology. Published by Elsevi er Science Inc.