Adult HSC compartment responds to multiple cytokine stimuli and expresses cytokine receptors. (A) Representative flow cytometric plot of phosphorylation responses [Stat3(pY705), Stat5(pY694)] after 15-minute stimulation with the indicated cytokines in healthy adult BM HSCs. HSCs are CD34⁺CD38⁻CD90⁺CD45RA⁻. The percentage of cells responding via each node is indicated in each quadrant and is calculated relative to the unstimulated control for each sample. Stimuli used were G-CSF (20 ng/mL), GM-CSF (20 ng/mL), FLT3 ligand (FL; 100 ng/mL), SCF (100 ng/mL), thrombopoietin (Tpo; 50 ng/mL), IL-3 (20 ng/mL), IL-6 (20 ng/mL), and a combination of FL, SCF, Tpo, IL-3, and IL-6 (referred to as FST36). Gray arrows represent directionality of response, but in the case where multiple nodes are activated, not necessarily the sequence of activation. (B) Heatmap of the average percentage of cells responding in each stem and progenitor compartment in normal adult BM after 15-minute cytokine stimulation. 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⁻. Measurements were obtained from 2-4 distinct biologic samples. Epo is erythropoietin, and 3 units/mL was used to stimulate cells. (C) Representative flow cytometric analyses of cytokine receptor expression on adult marrow HSCs. Red histogram is isotype control, blue histogram is cytokine receptor. Median fluorescence intensity (MFI) is indicated with isotype control on top. Measurements were taken on 3 distinct biologic samples.

Lack of CD114 expression distinguishes bona fide HSCs from transiently reconstituting multilineage progenitors. The percentage of human engraftment (determined by expression of human CD45) in the BM of NOD/SCID/IL-2Rγ^null (NSG) mice that received a transplantation of either CD114^neg/lo or CD114^pos subfractions of the human HSC compartment is indicated at (A) 6 weeks, or (B) 14 weeks after transplantation. Engrafted mice are defined as greater than 0.1% human chimerism of BMMCs. Black line represents the average percent human chimerism in each group of mice. The difference in the level of engraftment at week 14 is statistically significant (P = .001, 2-tailed Mann-Whitney test).

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.

G-CSF drives proliferation and cell-cycle entry within the HSC compartment. (A) Representative time-lapse images of prospectively sorted HSCs on hydrogel microwells at the beginning of acquisition (t = 24 hour) and final time point (t = 80 hour). Proliferation of single HSCs was tracked over time with some cells undergoing no divisions (red) and others dividing (blue). (B) Frequency of proliferation (≥ 1 cell division) for prospectively sorted, single HSCs plated on hydrogel microwells in serum-free, defined media supplemented with FL and SCF (FS) alone or in combination with 40 ng/mL G-CSF. Differences between proliferation frequency for CB (black) and normal BM (gray) at 80 and 92 hours, respectively, were statistically significant (P = .0004, paired, 1-tailed t test). (C) Representative flow cytometric detection of DNA (Hoechst 33342) and RNA (Pyronin Y) in prospectively sorted HSCs cultured on hydrogel substrate for 68 hours with gates corresponding to cell-cycle phase. (D) Frequency of cells in active cycle (ie, G1-S-G2-M) after 60-68 hours of culture with FS or FS+G. Difference in frequency of cells in active cycle is statistically significant (P = .0085, paired, 1-tailed t test).