Abstract
Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production.
Highlights
Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), residing at the top of the hematopoietic hierarchy, are responsible for the maintenance of steady-state and stress-adapted blood cell generation
Cells were activated with LPS and Pam3CSK4, a combination of Toll-like receptors (TLRs) ligands (TLRLs) that was reported [13] to effectively stimulate LSK cells to produce cytokines, including granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-6, and tumor necrosis factor-a (TNF-a) (Supplemental Figure 1)
The combination of PMA, ION, and BFA (PIB combination) and, to a lesser extent, PMA plus MN (PM combination) effectively maintained all three cytokines in activated mouse LSK cells (Supplemental Figure 2). Besides this acute infection model, similar results were obtained from a tumor-related model in which LSK cells were exposed to the culture supernatant of splenic stromal cells isolated from hepatoma-bearing mice (Hepa Splenic stromal cell supernatant (SPSC-SN); Supplemental Figure 3)
Summary
Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), residing at the top of the hematopoietic hierarchy, are responsible for the maintenance of steady-state and stress-adapted blood cell generation. Accumulating evidence suggests that HSPCs, like their mature myeloid and lymphoid descendants, are capable of translating danger signals into a plethora of cytokines that act in an autocrine or paracrine manner to regulate stress-induced hematopoiesis [13, 14] These cytokines represent a crucial line of communication and a way to amplify signals to convey the presence of danger among the HSPC community, along with information on what cell types should be produced in response to the threat. In 2014, David Baltimore and colleagues introduced a microfluidic-based platform to quantify secreted proteins at the single-cell level Using this technique, they found that the cytokine production ability of HSPCs exceeds that of mature myeloid and lymphoid cells in terms of the magnitude and the speed and breadth [13]. The methodological complexity, high cost, and relatively low cell throughput of this method have limited its widespread application, and single-cell, protein-level evidence in relevant studies is still mostly inferred or omitted
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