Single-cell STAT5 signal transduction profiling in normal and leukemic stem and progenitor cell populations reveals highly distinct cytokine responses.
BACKGROUND
Signal 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.


RESULTS
We 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
In 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.
Han
Lina
L
Department of Hematology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Wierenga
Albertus T J
AT
Rozenveld-Geugien
Marjan
M
van de Lande
Kim
K
Vellenga
Edo
E
Schuringa
Jan Jacob
JJ
D051997:ADP-ribosyl Cyclase 1 / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis; D019070:Cell Lineage; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D010766:Phosphorylation; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*
D018952:Antigens, CD34; D016207:Cytokines; D050799:STAT5 Transcription Factor; D051997:ADP-ribosyl Cyclase 1