Rapid activation of hematopoietic stem cells

Abstract

Adult hematopoietic stem cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation. Surprisingly, little is known about the earliest stages of HSCs activation. We utilize surface markers of HSCs activation, CD69 and CD317, revealing a response as early as 2 h after stimulation. The dynamic expression of HSCs activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify dose response, revealing a low threshold, and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence. Our data show that the response of adult stem cells to immune stimulation is rapid and sensitive, rapidly leading HSCs out of quiescence.

Keywords: Activation markers; BST2; CD317; CD69; Differentiation; HSC; Hematopoietic stem cells; Ki67; Proliferation; Rapid activation.

Fig. 1

Immune activation of hematopoietic stem and progenitor cells is rapid. A Schematic representation of the experimental settings involving injection of pIpC at different time points (0, 2, 4, and 24 h). B Representative FACS plots showing the percentage of CD69⁺ and CD317⁺ cells in the LSK (left panels) and LSKCD48⁻150⁺ HSCs (right panels) populations from control and pIpC stimulated mice at different time points. C, D Quantification of CD69 and CD317 expression in the LSK and LSKCD48⁻150⁺ HSCs populations in the BM of control and pIpC stimulated mice. E Schematic representation of the experimental settings involving injection of LPS at different time points (0, 2, 4, and 24 h). F Representative FACS plots showing the percentage of CD69⁺ and CD317⁺ cells in the LSK (left panels) and LSKCD48⁻150⁺ HSCs (right panels) populations from control and LPS-stimulated mice at different time points. G, H Quantification of CD69 and CD317 expression in the LSK and LSKCD48⁻150⁺ HSCs populations in the BM of control and LPS-stimulated mice. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

CD317 reveals a dose–response to pIpC above a minimal threshold. A Representative FACS plots showing the percentage of CD317⁺ cells in the LSK (upper panels) and LSKCD48⁻150⁺ HSCs (lower panels) populations from the BM of PBS-treated (control) and pIpC- stimulated mice under various pIpC doses (0.01–100 µg as positive control) at 24 h post-injection. B, C Quantification of the LSK CD317⁺ and LSKCD48⁻150⁺ HSCs CD317⁺ cell populations from the BM of PBS-treated (control) mice and mice treated with different doses of pIpC. D, E Linear regression model fitting the dose-dependent response with the expression of CD317 in LSK and LSKCD48⁻150⁺ HSCs populations. Solid lines represent the linear fit of data. Dotted lines represent 95% confidence intervals. Data are presented as average ± SD, a summary of three independent experiments, n ≥ 3 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

CD69 elevation is dose-dependent at higher concentrations of LPS. A Representative FACS plots showing the percentage of CD69⁺ cells in the LSK (upper panels) and LSKCD48⁻150⁺ HSCs (lower panels) populations from the BM of PBS-treated (control) mice and mice stimulated with various doses of LPS (0.0001–20 µg) at 2 h post-injection. B, C Quantification of the LSK CD69⁺ and LSKCD48⁻150⁺ HSCs CD69⁺ cell populations from the BM of PBS-treated (control) mice and mice treated with different doses of LPS. D, E Linear regression model fitting the dose-dependent response with the expression of CD69 in LSK and LSKCD48⁻150⁺ HSCs populations. Solid lines represent the linear fit of data. Dotted lines represent 95% confidence intervals. Data are presented as average ± SD, a summary of three independent experiments, n ≥ 3 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

CD69 and CD317 expression on activated stem and progenitor populations positively correlate with exit from quiescence. A Representative FACS plots showing cell cycle analysis using DAPI and intracellular expression of Ki67 in LSK CD317⁻ (left panel) and LSK CD317⁺ (right panel) cells from pIpC-stimulated mice at 4 and 24 h after injection. B, C Quantification of cell cycle phases (G0, G1, S, and M) in CD317⁻ and CD317⁺ fractions of the LSK and LSKCD48⁻150⁺ HSCs populations from pIpC-stimulated mice at 4 h and 24 h post-injection. D Representative FACS plots showing cell cycle analysis using DAPI and intracellular expression of Ki67 in LSK CD69⁻ (left panel) and LSK CD69⁺ (right panel) cells from LPS-stimulated mice at 2 and 24 h after injection. E, F Quantification of cell cycle phases (G0, G1, S, and M) in CD69⁻ and CD69⁺ fractions of the LSK and LSKCD48⁻ CD150⁺ HSCs populations from LPS-stimulated mice at 2 h and 24 h post-injection. Data are presented as average ± SD, a summary of three independent experiments, n ≥ 3 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001