Abstract
BackgroundSignal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. However, the manner in which STAT5 responds to early-acting and lineage-restricted cytokines, particularly in leukemic stem/progenitor cells, is largely unknown.Methodology/Principal FindingsWe optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at a single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO- and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma.Conclusions/SignificanceIn conclusion, our optimized multiparametric flow cytometry protocols allow the analysis of signal transduction at the single cell level in normal and leukemic stem and progenitor cells. Our study demonstrates highly distinctive cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.
Highlights
Hematopoiesis is largely regulated by signaling cascades that are activated by a wide variety of cytokines [1]
Various tested antibodies were insufficient in separating hematopoietic stem cells (HSCs), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) under F/M-treated conditions, but we were able to identify a set of antibodies with which the various compartments could be visualized by FACS under F/M-treated conditions (Fig. S3)
Various studies have shown that Signal Transducer and Activator of Transcription 5 (STAT5) can be activated by a plethora of cytokines, including FLT3 Ligand (FL), Stem Cell Factor (SCF), TPO, IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colonystimulating factor (GM-CSF) and EPO, subsequently affecting the self-renewal and hematopoietic differentiation program, less has been revealed about how the STAT5 signaling pathway responds to cytokine stimulation within the rare stem cells, or hematopoietic progenitor subfractions
Summary
Hematopoiesis is largely regulated by signaling cascades that are activated by a wide variety of cytokines [1]. The signals that emanate from cytokine receptors are translated into specific cellular responses via activation of transcription factors that induce expression of unique sets of target genes. One family of such transcription factors is the Signal Transducer and Activator of Transcription (STAT) family, which consists of 7 members, STAT1-6 whereby STAT5A and STAT5B are encoded by two separate genes. Loss-offunction studies demonstrated that long-term repopulating activity of hematopoietic stem cells (HSC) was impaired in STAT5Adeficient HSCs [5,6,7]. The manner in which STAT5 responds to early-acting and lineage-restricted cytokines, in leukemic stem/progenitor cells, is largely unknown
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