Characterization of cytokine interactions by flow cytometry and factorial analysis

Background: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activit y of other signaling pathways. These interactions are quantified using fact orial experimental design and analysis. Methods: Human umbilical cord blood (HUCB) CD34(+) cells were cultured in f ully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(IV)(7-3)) Or full factorial (2(4) ) design experiments. The cytokines studied were stem cell factor (SCF), in terleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granu locyte-colony stimulating factor (G-CSF), Fit-3 ligand, IL-6, IL-11, and er ythropoietin (EPO). In vitro cell divisions were tracked by staining CD34() cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, fo llowed by now cytometric analysis at 4 days of culture. In separate experim ents, lineage commitment and differentiation were determined at 7 days by i mmunophenotype. Results: In addition to the main effects of single cytokines, cytokine inte ractions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on eryt hroid and megakaryocytic development. The effect of Fit-3 ligand and SCF fa ctor on CD34(+) cell production was also less than additive, although the r esponse to both cytokines was greater than single cytokines. The only posit ive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. Conclusions: Factorial analysis provides a powerful methodology to study th e integration of multiple signals at the cellular and molecular level. Cyom etry 43:69-81, 2001. (C) 2001 Wiley-Liss, Inc.