Intracellular analysis of human HSPCs reveals a cellular subset within the HSC compartment that directly responds to G-CSF. (A) Simplified hematopoietic hierarchy. Self-renewing HSCs give rise to nonself-renewing multipotent progenitors (MPPs). MPPs give rise to common lymphoid (not pictured) or common myeloid progenitors (CMPs), which give rise to either granulocyte-macrophage restricted or megakaryocyte-erythroid restricted progenitors (GMPs and MEPs, respectively). These cells give rise to all mature myeloid lineages. Phenotypic definitions of subsets (all negative for mature lineage markers) are as follows: HSCs are CD34⁺CD38⁻CD90⁺CD45RA⁻, MPPs are CD34⁺CD38⁻CD90⁻CD45RA⁻, CMPs are CD34⁺CD38⁺CD123⁺CD45RA⁻, GMPs are CD34⁺CD38⁺CD123⁺CD45RA⁺, and MEPs are CD34⁺CD38⁺CD123^lo/−CD45RA⁻. (B) Experimental scheme for detection of intracellular signaling events in multiple hematopoietic stem and progenitor populations. See “Stimulation of HSPCs, resolution of HSCs, and phospho-signaling analysis” for details. (C) Flow cytometric analysis of lineage depleted CB progenitors stained for surface markers that identify hematopoietic subsets. Analysis of (i) live cells or (ii) cells after fixation-permeabilization. (iii) Flow cytometric analysis of Stat3(pY705) levels of prospectively sorted, CB HSCs stimulated for 15 minutes with G-CSF. (iv) Flow cytometric analysis of Stat3(pY705) levels in CB HSCs (same samples as panel iii) retrospectively gated after 15-minute stimulation of total lineage-negative progenitors with G-CSF.