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Several lines of evidence support the hypothesis that pluripotent stem cells (PSCs) reside in human tissues. Recently, we identified a population of very small embryonic-like (VSEL) SCs in umbilical cord blood (CB) (Leukemia2007;21:297-303). These VSELs are: i) very small in size (<6 um); ii) SSEA-4+/Oct-4+/CD133+/CD34+/CXCR4+/Linneg/ CD45neg; iii) responsive to a stromal derived factor (SDF)-1 gradient; and iv) possess large nuclei that contain primitive euchromatin. In the current study, we optimized their isolation/purification strategy and employed several imaging and molecular techniques to better analyze these primitive cells. We noticed that because of their small size, CD133+/ Linneg/CD45neg VSELs are lost (42.5±12.6%) during routine CB unit processing by volume depletion before storage/freezing. Interestingly, these cells are more resistant to changes following freezing and thawing as compared to normal hematopoietic (H)SCs. Interestingly, 82.7±17.3% of the initially frozen CD133+/Linneg/CD45neg VSELs are preserved in frozen CB units, while only 65.0±6.1% CD133+/Linneg/CD45neg HSCs are recovered. Furthermore, when we employed Ficoll centrifugation to purify CB mononuclear cells (CB MNCs), we found that while 59.8±7.2% of CD133+/Linneg/CD45neg VSELs were lost, their hematopoietic counterparts (CD133+/Linneg/CD45+) were almost fully recovered (Fig. 1A). These data indicate that other more “VSEL-saving” strategies of erythrocyte depletion should be developed because of the unusual size and density of these cells. We also established that the most the optimal “VSEL-saving” strategy to deplete erythrocytes from CB was hypotonic lysis. However, we noticed that during this procedure, lyzed erythrocytes release phosphatidyloserine positive (PS+) membrane-derived microvesicles (MVs) and these PS+ MVs preferentially bind to VSELs. Because of this phenomenon, VSELs become PS+ and may be falsely recognized as apoptotic cells in the Annexin-V-binding assay. The unique morphological features of VSELs were confirmed by several complementary imaging methods. ImageStream analysis revealed that VSELs are smaller than erythrocytes, are larger than platelets, and posses a high nuclear/cytoplasmic ratio (Fig. 1B). The fraction of CD133+/Linneg/CD45neg) VSELs with the smallest size (<6 um) exhibit a high cytoplasmic nuclear ratio and highly express Oct-4 in the nucleus and SSEA-4 and CD133 antigens on the surface. Finally, we found 2 to 3 times higher numbers of VSELs in CB samples from vaginal deliveries as compared to scheduled C-sections. This supports the idea that VSEL are released into CB due to delivery-related stress/hypoxia. In conclusion, CB contains a population of VSELs but ~50% of these cells are not recovered by currently employed volume-reduction strategies because of their unique morphology. Taking into consideration that VSELs may be employed in regenerative medicine, novel volume reduction/erythrocyte depletion strategies require development in CB banking to avoid loss of these rare, primitive, and important cells. Figure Figure

Proliferation, metabolism, and migration of hematopoietic stem/progenitor cells (HSPCs) are coordinated by receptors expressed on outer cell membranes that are integrated into microdomains, known as membrane lipid rafts (MLRs). These structures float freely in the cell membrane bilayer and are enriched in cholesterol and sphingolipids for their functional integrity. Receptors, if expressed in MLRs, have prolonged occupancy on the cell surface and enhanced signaling power. Based on this, we have become interested in the regulation of synthesis of MLRs components in HSPCs. To address this, we tested the effect of selected factors that promote proliferation or migration and their potential involvement in the synthesis of MLRs components in HSPCs. Based on our previous research showing that HSPCs from Nox2-KO and Nlrp3-KO mice display a profound defect in MLRs formation, we focused on the role of Nox2-ROS-Nlrp3 inflammasome in regulating lipogenesis in HSPCs. We found that while at steady state conditions, Nox2-derived ROS is required for a proper expression of enzymes regulating lipogenesis, during inflammation, this effect is augmented by Nlrp3 inflammasome. Thus, our data sheds new light on the regulation of lipogenesis in HSPCs and the involvement of the Nox2-ROS-Nlrp3 inflammasome axis that differently regulates lipogenesis at steady state conditions and in response to inflammation, modulating MLRs-mediated responsiveness of these cells to external stimuli. Graphical Abstract

Hematopoietic and immune cells originate from a common hematopoietic/lymphopoietic stem cell what explains that these different cell types often share the same receptors and respond to similar factors. Moreover, the common goal of both lineages is to ensure tissue homeostasis under steady-state conditions, fight invading pathogens, and promote tissue repair. We will highlight accumulating evidence that innate and adaptive immunity modulate several aspects of hematopoiesis within the hormetic zone in which the biological response to low exposure to potential stressors generally is favorable and benefits hematopoietic stem/progenitor cells (HSPCs). Innate immunity impact on hematopoiesis is pleiotropic and involves both the cellular arm, comprised of innate immunity cells, and the soluble arm, whose major component is the complement cascade (ComC). In addition, several mediators released by innate immunity cells, including inflammatory cytokines and small antimicrobial cationic peptides, affect hematopoiesis. There are intriguing observations that HSPCs and immune cells share several cell-surface pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and cytosol-expressed NOD, NOD-like, and RIG-I-like receptors and thus can be considered “pathogen sensors”. In addition, not only lymphocytes but also HSPCs express functional intracellular complement proteins, defined as complosome which poses challenging questions for further investigation of the intracellular ComC-mediated intracrine regulation of hematopoiesis.

Background . The SARS-CoV-2 pandemic, with its high mortality, has become an urgent clinical problem. It is well established that SARS-CoV-2 enters human cells after binding to the angiotensin-converting enzyme 2 (ACE2) receptor and utilizes a spike protein (S) for attachment and entry into the cells, leading to their lysis or damage. This infection damages several organs, including lungs, heart, blood vessels, kidneys, and intestines and may lead to a fatal complication known as a "cytokine storm", which is the result of uncontrolled hyperactivation of the innate immunity-initiated response and the release of several pro-inflammatory cytokines. An important potential aspect of SARS-CoV-2 infection is damage to the stem cell compartment, which may lead to severe complications from the infection. The ACE2 receptor has been described as being expressed on the surfaces of certain types of stem cells, including specified hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). These cells could be potentially damaged and lysed after virus entry or could undergo pyroptosis due to hyperactivation of Nlrp3 inflammasomes. Supporting this possibility, we recently reported that all Nlrp3 inflammasome components are expressed in HSCs (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020).https://doi.org/10.1007/s12015-020-10005-w). Hypothesis. We hypothesized that one of the triggers of a cytokine storm could be interaction of the ACE2 receptor with the SARS-CoV-2 spike protein, leading to hyperactivation of Nlrp3 inflammsomes in target cells including population of stem cells.Materials and Methods. Experiments were performed on human stem cells at different levels of specification, including very small CD34+CD133+lin-CD45- cells, which may become specified into HSCs and EPCs, as well as human CD34+Lin-CD45+ HSCs and CD34+ CD133+ KDR+ CD31+ EPCs. These cells were phenotyped for expression of ACE2 and the SARS-CoV-2 entry-facilitating transmembrane protease TMPRSS2 at the mRNA level and by FACS at the protein level. Next, we exposed these cells to the NCP-CoV (2019-nCoV) spike protein (S1+S2 ECD, expressed with a His-tag; Sino Biological) at a concentration of 10 nM. After 16 h of incubation, the cells were lysed, and total RNA was isolated for qRT-PCR analysis of Nlrp3 and essential Nlrp3 inflammasome components, including ASC, caspase 1, IL1b, and IL18. In some experiments, UCB-derived HSCs were plated into 96-well plates and stimulated with NCP-CoV (2019-nCoV) spike protein, as described above, alone or together with angiotensin 1-7 or the anti-inflammatory heme oxygenase 1 (HO-1) activator CoPP. Results. First, we observed that the ACE2 receptor and SARS-CoV-2 entry-facilitating transmembrane protease TMPRSS2 are expressed by all types of stem cells evaluated in our studies. Moreover, we detected activation of Nlrp3 inflammasomes in response to viral spike protein. This activation was inhibited by exposure of the stimulated cells to angiotensin 1-7 or CoPP. Conclusions. We envision that, in addition to directly infecting target cells, virus can hyperactivate the Nlrp3 inflammasome in stem cells, which may trigger their pyroptosis. Therefore, since we still do not have an effective SARS-CoV-2 vaccine in hand, the results presented in our current work suggest that inhibition of the Nlrp3 inflammasome by the small-molecule inhibitor MCC950 or application of Nlrp3 inflammasome inhibitors, such as Ang (1-7) or heme oxygenase 1 activators, could find potential clinical application to prevent the onset of a cytokine storm and cell pyroptosis. Disclosures No relevant conflicts of interest to declare.

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We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway and, as result of this, several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood2003;101,3784; Blood2004;103,2071; Blood2005;105,40). Bioactive CC anaphylatoxins (C5a and desArgC5a) are also potent chemoattractants of granulocytes that bind to G-protein-coupled, seven trans-membrane span C5a receptors (C5aR and C5L2) on these cells. To learn more on the role of C5 cleavage fragments in HSPC mobilization, we studied mobilization in C5−/− and C5aR−/− mice as well as their normal wildtype littermates. Mobilization was induced by granulocyte colony-stimulating factor (G-CSF; high 250 μg/kg/6 days and low dose 50 μg/kg/6 days) or zymosan (20 mg/1kg/1 hour), which activate classical and alternative pathways of CC, respectively. We evaluated mobilization efficiency by counting the number of SKL cells, colony-forming unit granulocyte-macrophages (CFU-GMs), and white blood cells circulating in peripheral blood. In parallel, we employed transmission electron microscopy (TEM) to study the morphology and integrity of BM vessels in the BM-blood barrier. Activation of CC was measured by ELISA for C3 cleavage fragments and by histochemical staining for membrane attack-complex (MAC) depositions in BM tissue. We found by ELISA and histochemistry that CC activation correlates with the level of HSPC mobilization in wildtype mice and that mobilization of HSPCs was always preceded by the release of granulocytes from BM. Thus, granulocytes are the first wave of cells that increase in number during mobilization in peripheral blood. Mobilization studies in C5−/− revealed that these animals are very poor mobilizers. TEM studies demonstrated that hematopoietic cells together with granulocytes accumulated around small vessels in the BM of C5−/− animals, but they did not migrate or cross the BM-endothelial barrier. Since C5 cleavage fragments C5a and desArgC5a are potent chemoatrractants for granulocytes but not HSPCs, we hypothesize that a lack of both these anaphylatoxins in C5−/− animals prevents egress of granulocytes from BM, which always precedes egress of HSPCs. Furthermore, in C5aR−/−, mice mobilization was normal after administration of a high optimal dose of G-CSF. However, mobilization was significantly lower after a suboptimal dose of G-CSF or administration of zymosan. This indicates that another alternative receptor for C5a and desArgC5a (C5L2) may compensate for C5aR deficiency and that it plays a role in the egress of granulocytes from the BM as well. Thus, this study demonstrates that cells from the granulocytic lineage are actively involved in mobilization in a C5a,-desArgC5a-C5aR manner not only by secreting proteases that create a proteoytic environment in BM, but also as a kind of “ice-breaker” type cells necessary for disintegration of the endothelial-BM barrier to enable HSPCs to egress from the BM microenvironment. In cases of granulocytopenia or if granulocytes are not mobilized as seen in C5−/− mutants, mobilization of HSPCs is very poor. Thus, modulation of CC activation in the BM and stimulation of granulocyte egress from the BM into circulation may help to develop more efficient strategies for HSPC mobilization.

Purpose of review Hematopoiesis is co-regulated by innate immunity, which is an ancient evolutionary defense mechanism also involved in the development and regeneration of damaged tissues. This review seeks to shed more light on the workings of the Nlrp3 inflammasome, which is an intracellular innate immunity pattern recognition receptor and sensor of changes in the hematopoietic microenvironment, and focus on its role in hematopoieisis. Recent findings Hematopoietic stem progenitor cells (HSPCs) are exposed to several external mediators of innate immunity. Moreover, since hemato/lymphopoietic cells develop from a common stem cell, their behavior and fate are coregulated by intracellular innate immunity pathways. Therefore, the Nlrp3 inflammasome is functional both in immune cells and in HSPCs and affects hematopoiesis in either a positive or negative way, depending on its activity level. Specifically, while a physiological level of activation regulates the trafficking of HSPCs and most likely maintains their pool in the bone marrow, hyperactivation may lead to irreversible cell damage by pyroptosis and HSPC senescence and contribute to the origination of myelodysplasia and hematopoietic malignancies. Summary Modulation of the level of Nrp3 inflammasome activation will enable improvements in HSPC mobilization, homing, and engraftment strategies. It may also control pathological activation of this protein complex during HSPC senescence, graft-versus-host disease, the induction of cytokine storms, and the development of hematopoietic malignancies.