Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma

M. Guo, W. M. Miller, E. T. Papoutsakis, S. Patel, C. James, C. Goolsby, J. N. Winter*

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Background: Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34+-selected PBMC, have shown no advantage for CD34+ cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. Methods: Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. Results: The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34+ cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Discussion: Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.

Original languageEnglish (US)
Pages (from-to)183-194
Number of pages12
JournalCytotherapy
Volume1
Issue number3
DOIs
StatePublished - Jan 1 1999

Keywords

  • Ex vivo expansion
  • Stem cell transplantation

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Genetics(clinical)
  • Cell Biology
  • Cancer Research
  • Transplantation

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