Use of fractional factorial analysis and cell tracking to determine the optimal cytokine combinations for the production and survival of primitive and committed progeny from cord blood stem cells

J. Case, A. Rice

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

An optimal cytokine combination for the production of primitive and committed progeny from cord blood (CB) has yet to be defined. We have designed two-level fractional factorial experiments (27-3iv) to determine: 1) the main factors which have either positive or negative interactions with G-CSF or MGDF, 2) two-factor combinations which have either positive or negative interactions with G-CSF or MGDF and 3) factors which amplify both granulocytic and megakaryocytic cell production. The intracellular tracking dye. 5-(-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) was used concurrently to determine the number of cell divisions that had occurred after the cells had been exposed to cytokines. MACS enriched, flow sorted CD34 CB cells were incubated for 7 days in serum free media with various combinations of cytokines (G-CSF, FL. SCF, IL-3, MGDF, IL-11, IL-6 and Epo). The immunophenotype of cells before and after cytokine incubation was determined by three-colour flow cytometry using a panel of monoclonal antibodies to distinguish between committed and primitive progenitors (CD41 FITC/CD71 PE/CD33 PE-Cy5, CD61 FITC/CD38 PE/CD34 PE-Cy5). In separate experiments cell survival and divisional recruitment (at least one cell division) was determined from the retention of CFSE at 4 days of culture. Factors which amplify both granulocytic and megakaryocytic as well as CD34 38" cell production were FL (p<0.001), SCF (p<0.001) and MGDF (rxO.Ol). SCF, IL-3 and FL were shown to be the main factors that promote cell survival and divisional recruitment of CB CD34+ cells. Importantly, no single factors were shown to inhibit the generation of these cell types. In conclusion, it is likely that the cytokines which amplify production of both myeloid and megakaryocytic cells (FL, SCF and MGDF) act on uncommitted progenitors. Factorial experimental design analysis has provided an appropriate statistical model for understanding the complex nature of cytokine interactions that regulate in vitro production of hematopoietic cell progeny.

Original languageEnglish (US)
Number of pages1
JournalExperimental Hematology
Volume26
Issue number8
StatePublished - Dec 1 1998
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

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