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


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 1.5x10(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).


CONCLUSIONS
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.
Scheding
S
S
Department of Internal Medicine, Division of Hematology, Oncology, Rheumatology and Immunology, University of Tübingen, Tübingen, Germany. stefan.scheding@t-online.de
Meister
B
B
Bühring
H J
HJ
Baum
C M
CM
Mc Kearn
J P
JP
Bock
T
T
Kanz
L
L
Brugger
W
W
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology*; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology / Q000031:analogs & derivatives; D015850:Interleukin-6 / Q000494:pharmacology; D008565:Membrane Proteins / Q000494:pharmacology; D010446:Peptide Fragments; D010455:Peptides / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D015703:Antigens, CD; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D007375:Interleukin-1; D007377:Interleukin-3; D015850:Interleukin-6; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; C093340:daniplestim; D016178:Granulocyte-Macrophage Colony-Stimulating Factor