Distinct signaling programs control human hematopoietic stem cell survival and proliferation

Several growth factors (GFs) that together promote quiescent humanhematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which theseGFsregulate the survival, proliferation. and differentiation ofhumanHSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34(+) cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with aCD34(+) CD38(-) CD45RA CD90(+) CD49f(+) (CD49f(+)) phenotype (similar to 10% HSCswith > 6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and b-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f(+) cells, different signaling intermediates correlated with the levels of the myeloid-and lymphoidassociated transcription factors measured. Phenotypically similar, but CD90(-) CD49f(-)cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90(+) CD49f(+) cells, but showed similar response amplitudes to the same GFs. Importantly, wefound activation or inhibition ofAKTand beta-catenin directly altered immediateCD49f (+) cell survivalandproliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.