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NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration., (© 2024. The Author(s).)

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries., (© 2024. The Author(s).)

A cell's most significant existential task is to survive by ensuring proper metabolism, avoiding harmful stimuli, and adapting to changing environments. It explains why early evolutionary primordial signals and pathways remained active and regulate cell and tissue integrity. This requires energy supply and a balanced redox state. To meet these requirements, the universal intracellular energy transporter purine nucleotide-adenosine triphosphate (ATP) became an important signaling molecule and precursor of purinergic signaling after being released into extracellular space. Similarly, ancient proteins involved in intracellular metabolism gave rise to the third protein component (C3) of the complement cascade (ComC), a soluble arm of innate immunity. These pathways induce cytosol reactive oxygen (ROS) and reactive nitrogen species (RNS) that regulate the redox state of the cells. While low levels of ROS and RNS promote cell growth and differentiation, supra-physiological concentrations can lead to cell damage by pyroptosis. This balance explains the impact of purinergic signaling and innate immunity on cell metabolism, organogenesis, and tissue development. Subsequently, along with evolution, new regulatory cues emerge in the form of growth factors, cytokines, chemokines, and bioactive lipids. However, their expression is still modulated by both primordial signaling pathways. This review will focus on the data that purinergic signaling and innate immunity carry on their ancient developmental task in hematopoiesis and specification of hematopoietic stem/progenitor cells (HSPCs). Moreover, recent evidence shows both these regulatory pathways operate in a paracrine manner and inside HSPCs at the autocrine level., (© 2024. The Author(s).)

Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide an evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bone marrow (BM) contains not only hematopoietic stem cells (HSCs) but also some very rare, early development, small quiescent stem cells that, upon activation, may differentiate across germlines. These small cells, named very small embryonic like stem cells (VSELs), can undergo specification into several types of cells including HSCs. Interestingly, murine BM is also home to a "mystery" population of small CD45 + stem cells with many of the phenotypic characteristics attributed to resting HSCs. Since the size of the "mystery" population cells are between that of VSELs and HSCs, and because CD45 - VSELs can be specified into CD45 + HSCs, we hypothesized that the quiescent CD45 + "mystery" population could be a missing developmental link between VSELs and HSCs. To support this hypothesis, we showed that VSELs first became enriched for HSCs after acquiring expression of the CD45 antigen already expressed on "mystery" stem cells. Moreover, VSELs freshly isolated from BM similar to the "mystery" population cells, are quiescent and do not reveal hematopoietic potential in in vitro and in vivo assays. However, we noticed that CD45 + "mystery" population cells, similar to CD45 - VSELs, became specified into HSCs after co-culture over OP9 stroma. We also found that mRNA for Oct-4, a pluripotency marker that is highly expressed in VSELs, is also detectable in the "mystery" population cells, albeit at a much lower level. Finally, we determined that the "mystery" population cells specified over OP9 stroma support were able to engraft and establish hematopoietic chimerism in lethally irradiated recipients. Based on these results, we propose that the murine BM "mystery" population could be an intermediate population between BM-residing VSELs and HSCs already specified for lympho-hematopoietic lineages., (© 2023. The Author(s).)

Hematopoietic stem/progenitor cells (HSPCs) express receptors for complement cascade (ComC) cleavage fragments C3a and C5a and may respond to inflammation-related cues by sensing pathogen-associated molecular pattern molecules (PAMPs) released by pathogens as well as non-infectious danger associated molecular pattern molecules (DAMPs) or alarmin generated during stress/tissue damage sterile inflammation. To facilitate this HSPCs are equipped with C3a and C5a receptors, C3aR and C5aR, respectively, and express on the outer cell membrane and in cytosol pattern recognition receptors (PPRs) that sense PAMPs and DAMPs. Overall, danger-sensing mechanisms in HSPCs mimic those seen in immune cells, which should not surprise as hematopoiesis and the immune system develop from the same common stem cell precursor. This review will focus on the role of ComC-derived C3a and C5a that trigger nitric oxide synthetase-2 (Nox2) complex to release reactive oxygen species (ROS) that activate important cytosolic PRRs-Nlrp3 inflammasome, which orchestrates responsiveness of HSPCs to stress. Moreover, recent data indicate that in addition to circulating in peripheral blood (PB) activated liver-derived ComC proteins, a similar role plays ComC expressed and intrinsically activated in HSPCs known as "complosome". We postulate that ComC triggered Nox2-ROS-Nlrp3 inflammasome responses, if they occur within non-toxic to cells' "hormetic range of activation", positively regulate HSCs migration, metabolism, and proliferation. This sheds a new light on the immune-metabolic regulation of hematopoiesis., (© 2023. The Author(s).)

Very small embryonic-like stem cells (VSELs) are a dormant population of development early stem cells deposited in adult tissues that as demonstrated contribute to tissue/organ repair and regeneration. We postulated developmental relationship of these cells to migrating primordial germ cells (PGCs) and explained the quiescent state of these cells by the erasure of differently methylated regions (DMRs) at some of the paternally imprinted genes involved in embryogenesis. Recently, we reported that VSELs began to proliferate and expand in vivo in murine bone marrow (BM) after exposure to nicotinamide (NAM) and selected pituitary and gonadal sex hormones. In the current report, we performed proteomic analysis of VSELs purified from murine bone marrow (BM) after repeated injections of NAM + Follicle-Stimulating Hormone (FSH) that in our previous studies turned out to be an effective combination to expand these cells. By employing the Gene Ontology (GO) resources, we have performed a combination of standard GO annotations (GO-CAM) to produce a network between BM steady-state conditions VSELs (SSC-VSELS) and FSH + NAM expanded VSELs (FSH + NAM VSELs). We have identified several GO biological processes regulating development, organogenesis, gene expression, signal transduction, Wnt signaling, insulin signaling, cytoskeleton organization, cell adhesion, inhibiting apoptosis, responses to extra- and intracellular stimuli, protein transport and stabilization, protein phosphorylation and ubiquitination, DNA repair, immune response, and regulation of circadian rhythm. We report that VSELs express a unique panel of proteins that only partially overlapped with the proteome of BM - derived hematopoietic stem cells (HSCs) and hematopoietic mononuclear cells (MNCs) and respond to FSH + NAM stimulation by expressing proteins involved in the development of all three germ layers. Thus, our current data supports further germ-lineage origin and multi germ layer differentiation potential of these cells., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

Evidence indicates that bone marrow (BM)-residing hematopoietic stem/progenitor cells (HSPCs) are released into peripheral blood (PB) after administration of pro-mobilizing drugs, which induce a state of sterile inflammation in the BM microenvironment. In the reverse process, as seen after hematopoietic transplantation, intravenously injected HSPCs home and engraft into BM niches. Here again, conditioning for transplantation by myeloablative chemo- or radiotherapy induces a state of sterile inflammation that promotes HSPC seeding to BM stem cell niches. Therefore, the trafficking of HSPCs and their progeny, including granulocytes and monocytes/macrophages, is regulated by a response to pro-inflammatory stimuli. This responsiveness to inflammatory cues is also preserved after malignant transformation of hematopoietic cells. Results from our laboratory indicate that the responsiveness of hematopoietic cells to pro-inflammatory stimuli is orchestrated by Nlrp3 inflammasome. As reported, HO-1 effectively attenuates intracellular activation of Nlrp3 inflammasome as well as the pro-inflammatory effects of several humoral mediators, including complement cascade (ComC) cleavage fragments that promote migration of hematopoietic cells. Based on this finding, inhibition of HO-1 activity may become a practical strategy to enhance the mobilization and homing of normal HSPCs, and, alternatively, its activation may prevent unwanted spread and in vivo expansion of leukemic cells. Graphical Abstract.

Angiotensin-converting enzyme 2 (ACE2) plays an important role as a member of the renin-angiotensin-aldosterone system (RAAS) in regulating the conversion of angiotensin II (Ang II) into angiotensin (1-7) (Ang [1-7]). But at the same time, while expressed on the surface of human cells, ACE2 is the entry receptor for SARS-CoV-2. Expression of this receptor has been described in several types of cells, including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which raises a concern that the virus may infect and damage the stem cell compartment. We demonstrate for the first time that ACE2 and the entry-facilitating transmembrane protease TMPRSS2 are expressed on very small CD133 + CD34 + Lin - CD45 - cells in human umbilical cord blood (UCB), which can be specified into functional HSCs and EPCs. The existence of these cells known as very small embryonic-like stem cells (VSELs) has been confirmed by several laboratories, and some of them may correspond to putative postnatal hemangioblasts. Moreover, we demonstrate for the first time that, in human VSELs and HSCs, the interaction of the ACE2 receptor with the SARS-CoV-2 spike protein activates the Nlrp3 inflammasome, which if hyperactivated may lead to cell death by pyroptosis. Based on this finding, there is a possibility that human VSELs residing in adult tissues could be damaged by SARS-CoV-2, with remote effects on tissue/organ regeneration. We also report that ACE2 is expressed on the surface of murine bone marrow-derived VSELs and HSCs, although it is known that murine cells are not infected by SARS-CoV-2. Finally, human and murine VSELs express several RAAS genes, which sheds new light on the role of these genes in the specification of early-development stem cells. Graphical Abstract •Human VSELs and HSCs express ACE2 receptor for SARS-CoV2 entry. •Interaction of viral spike protein with ACE2 receptor may hyperactivate Nlrp3 inflammasome which induces cell death by pyroptosis. •SARS-CoV2 may also enter cells and eliminate them by cell lysis. •What is not shown since these cells express also Ang II receptor they may hyperactivate Nlrp3 inflammasome in response to Ang II which may induce pyroptosis. Our data indicates that Ang 1-7 may have a protective effect.

Fast and efficient homing and engraftment of hematopoietic stem progenitor cells (HSPCs) is crucial for positive clinical outcomes from transplantation. We found that this process depends on activation of the Nlrp3 inflammasome, both in the HSPCs to be transplanted and in the cells in the recipient bone marrow (BM) microenvironment. For the first time we provide evidence that functional deficiency in the Nlrp3 inflammasome in transplanted cells or in the host microenvironment leads to defective homing and engraftment. At the molecular level, functional deficiency of the Nlrp3 inflammasome in HSPCs leads to their defective migration in response to the major BM homing chemoattractant stromal-derived factor 1 (SDF-1) and to other supportive chemoattractants, including sphingosine-1-phosphate (S1P) and extracellular adenosine triphosphate (eATP). We report that activation of the Nlrp3 inflammasome increases autocrine release of eATP, which promotes incorporation of the CXCR4 receptor into membrane lipid rafts at the leading surface of migrating cells. On the other hand, a lack of Nlrp3 inflammasome expression in BM conditioned for transplantation leads to a decrease in expression of SDF-1 and danger-associated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor is a widely used anticonvulsant drug. VPA is also under clinical evaluation to be employed in anticancer therapy, as an antithrombotic agent or a molecule to be used in the stem cells expansion protocols. Since endothelial colony forming cells (ECFC) has been identified as the human postnatal vasculogenic cells involved in thrombotic disorders and serve as a promising source of immature cell for vascular repair, objectives of the present study were to determine how VPA contributes to ECFC commitment and their angiogenic properties. We examined the effect of VPA on ECFC obtained from cord blood by evaluating colony number, proliferation, migration and their sprouting ability in vitro, as well as their in vivo vasculogenic properties. VPA inhibited endothelial differentiation potential from of cord blood derived stem cells associated with decreased proliferation and sprouting activity of cultured ECFC. VPA treatment significantly decreased the vessel-forming ability of ECFC transplanted together with mesenchymal stem cells (MSC) in Matrigel implants in nude mice model. Surprisingly, a microscopic evaluation revealed that VPA induces marked morphological changes from a cobblestone-like EC morphology to enlarged spindle shaped morphology of ECFC. RT-qPCR and a CD31/CD90 flow cytometry analysis confirmed a phenotypic switch of VPA-treated ECFC to mesenchymal-like phenotype. In conclusion, the pan-HDAC inhibitor VPA described for expansion of hematopoietic stem cells and very small embryonic like stem cells cannot be successfully employed for differentiation of endothelial lineage committed ECFC into functional endothelial cells. Our data also suggest that VPA based therapeutics may induce endothelial dysfunction associated with fibrosis that might induce thrombosis recurrence or venous insufficiency.

We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.

Recent evidence indicates that the occurrence of psychiatric disorders in patients is linked to a local "sterile" inflammation of brain or due to a systemic inflammation process that affects the central nervous system. This is supported by the observation that in peripheral blood of psychotic patients are detectable several mediators and markers of inflammation as well as clinical data on correlations between systemic chronic inflammatory processes and psychiatric disorders. This may explain why some reported anti-inflammatory treatment strategies have beneficial effects on ameliorating psychotic events. In this review we will present a concept that aberrant purinergic signaling and increases in extracellular level of adenosine triphosphate (ATP) in the brain parenchyma may lead to activation of Nlrp3 inflammasome in microglia cells and as a consequence microglia released danger associated molecular pattern (DAMP) proteins activate complement cascade (ComC) in mannan binding lectin (MBL) - dependent manner. Activation of ATP-Nlrp3 inflammasome-ComC axis may also orchestrate trafficking of stem cells released from bone marrow into peripheral blood observed in psychotic patients. Based on this, the ATP-Nlrp3 inflammasome-ComC axis may become a target for new therapeutic approaches, which justifies the development and clinical application of efficient anti-inflammatory treatment strategies targeting this axis in psychiatry.

Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a "sterile inflammation" in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1β and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP-Nlrp3-ComC axis in the egress of stem cells into PB.

Background: Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), as well as very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue and organ injury (e.g., heart infarct or stroke)., Objective: We seek to determine whether these cells are also mobilized into PB in patients with skin burn injuries., Methods: Forty-four (44) patients (33-57 years of age) with total body surface burn area of 30-60%, as well as 23 healthy control subjects, were recruited and PB samples were harvested during the first 24 hours, day +2, and day +5 after burn injury and compared to normal controls. The circulating human CD34(+)CD133(+) cells enriched for HSPCs, as well as small CXCR4(+)CD34(+)CD133(+) subsets of Lin(-)CD45(-) cells that correspond to the population of VSELs, were counted by FACS and evaluated by direct immunofluorescence staining for pluripotency markers (Oct-4, Nanog, and SSEA-4). In parallel, we also measured by ELISA the serum concentration of factors that regulate stem cell trafficking, such as SDF-1, VEGF, and HGF., Results: Our data indicate that skin burn injury mobilizes cells expressing stem cell-associated markers, such as CD133, CD34, and CXCR4, into PB. More importantly, we found an increase in the number of circulating primitive, small Oct-4(+)Nanog(+)SSEA-4(+)CXCR4(+)lin(-)CD45(-) VSELs. All these changes were accompanied by increased serum concentrations of SDF-1 and VEGF., Limitations: Further studies are needed to fully assess the role of mobilized stem cells in the healing process to see if they can contribute to skin regeneration., Conclusion: Skin burn injury triggers the mobilization of HSPCs and CXCR4(+) VSELs, while the significance and precise role of mobilized VSELs in skin repair requires further study.

The morphology of several tumors mimics developmentally early tissues; tumors often express early developmental markers characteristic for the germ line lineage. Recently, our group identified a population of very small stem cells (SCs) in murine bone marrow (BM) and other adult organs that express several markers characteristic for epiblast/germ line-derived SCs. We named these rare cells "Very Small Embryonic/Epiblast-like Stem Cells (VSELs)." We hypothesized that these cells that express both epiblast and germ line markers are deposited during early gastrulation in developing tissues and organs and play an important role in the turnover of tissue-committed (TC) SCs. To support this, we envision that the germ line is not only the origin of SCs, but also remains as a scaffold or back-up for the SC compartment in adult life. Furthermore, we noticed that VSELs are protected from uncontrolled proliferation and teratoma formation by a unique DNA methylation pattern in some developmentally crucial imprinted genes, which show hypomethylation or erasure of imprints in paternally methylated genes and hypermethylation of imprints in the maternally methylated. In pathological situations, however, we hypothesize that VSELs could be involved in the development of several malignancies. Therefore, potential involvement of VSELs in cancerogenesis could support century-old concepts of embryonic rest- or germ line-origin hypotheses of cancer development. However, we are aware that this working hypothesis requires further direct experimental confirmation.