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.
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.
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 1.5x10(4)/mL in 21% O(2).
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).
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.