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We postulated that mobilization, homing, and engraftment of hematopoietic stem/progenitor cells (HSCPs) is facilitated by a state of sterile inflammation induced in bone marrow (BM) after administration of pro-mobilizing drugs or in response to pre-transplant myeloablative conditioning. An important role in this phenomenon plays purinergic signaling that by the release of extracellular adenosine triphosphate (eATP) activates in HSPCs and in cells in the hematopoietic microenvironment an intracellular pattern recognition receptor (PPR) known as Nlrp3 inflammasome. We reported recently that its deficiency results in defective trafficking of HSPCs. Moreover, it is known that eATP after release into extracellular space is processed by cell surface expressed ectonucleotidases CD39 and CD73 to extracellular adenosine (eAdo) that in contrast to eATP shows an anti-inflammatory effect. Based on data that the state of sterile inflammation promotes trafficking of HSPCs, and since eAdo is endowed with anti-inflammatory properties we become interested in how eAdo will affect the mobilization, homing, and engraftment of HSPCs and which of eAdo receptors are involved in these processes. As expected, eAdo impaired HSPCs trafficking and this occurred in autocrine- and paracrine-dependent manner by direct stimulation of these cells or by affecting cells in the BM microenvironment. We report herein for the first time that this defect is mediated by activation of the A2B receptor and a specific inhibitor of this receptor improves eAdo-aggravated trafficking of HSPCs. To explain this at the molecular level eAdo-A2B receptor interaction upregulates in HSPCs in NF-kB-, NRF2- and cAMP-dependent manner heme oxygenase-1 (HO-1), that is Nlrp3 inflammasome inhibitor. This corroborated with our analysis of proteomics signature in murine HSPCs exposed to eAdo that revealed that A2B inhibition promotes cell migration and proliferation. Based on this we postulate that blockage of A2B receptor may accelerate the mobilization of HSPCs as well as their hematopoietic reconstitution and this approach could be potentially considered in the future to be tested in the clinic. [ABSTRACT FROM AUTHOR]

Significance: The success rate of hematopoietic stem cell transplantation depends mainly on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) followed by the speed of their engraftment in the myeloablated transplant recipient. Therefore, clinical outcomes will significantly benefit from accelerating the homing and engraftment of these cells. This is, in particular, important when the number of cells available for the transplantation of HSPCs is limited. Recent Advances: We postulated that myeloablative conditioning for hematopoietic transplantation by radio- or chemotherapy induces a state of sterile inflammation in transplant recipient peripheral blood (PB) and bone marrow (BM). This state is mediated by activation of the BM stromal and innate immunity cells that survive myeloablative conditioning and respond to danger-associated molecular patterns released from the cells damaged by myeloablative conditioning. As a result of this, several factors are released that promote proper navigation of HSPCs infused into PB of transplant recipient and prime recipient BM to receive transplanted cells. Critical Issues: We will present data that cellular innate immunity arm and soluble arm comprised complement cascade proteins, promoting the induction of the BM sterile inflammation state that facilitates the navigation, homing, and engraftment of HSPCs. Future Directions: Deciphering these mechanisms would allow us to better understand the mechanisms that govern hematopoietic recovery after transplantation and, in parallel, provide important information on how to optimize this process in the clinic by employing small molecular modifiers of innate immunity and purinergic signaling. Antioxid. Redox Signal. 37, 1254–1265. [ABSTRACT FROM AUTHOR]

Simple Summary: A well-balanced diet seems to play a key role in disease prevention and health promotion in young animals. Therefore, many attempts have been made to supplement feeds with novel nutritional components, with potential prebiotic capacity. It seems that chicory root fulfils those criteria as it contains high amounts of inulin-type fructans. Hence, the aim of the study was to determine the effect of dietary supplementation with 4% dried chicory root on the cecal mucosa proteome of piglets. It is shown that this feed additive may affect cellular metabolism in the cecal epithelium and may be beneficial for gut health. Prebiotics are known to have many beneficial effects on intestinal health by modulating the gut microbiota composition, thereby affecting epithelial cell proliferation and metabolism. This study had two aims: (1) to identify the protein constituents in the cecal mucosa of 50-day-old healthy (PIC × Penarlan P76) barrows, and (2) to assess the effects of 4% inclusion of dried chicory root in a cereal-based diet on the cecal mucosa proteome changes. Pigs (eight per group) were randomly allotted to the groups and were fed a control diet from the tenth day of life (C) or a diet supplemented with 4% of died chicory root (CR), for 40 days. At the age of 50 days, animals were sacrificed and cecal tissue samples were collected. It was found that feeding a CR diet significantly decreased the expression of 16 cecal mucosa proteins. Among them, fifteen proteins were down-regulated, while only one (KRT20) was shown to be up-regulated when compared to the C group. Dietary supplementation with CR caused down-expression of metabolism-associated proteins including enzymes involved in the process of glycolysis (G6PD, TPI1, ALDH9A1, CKMT1 and AKR1A1) as well as those engaged in transcriptional and translational activity (PRPF19, EEF1G) and several structural proteins (ACTR3, KRT77, CAP1 and actin). From our findings, it is possible to conclude that dietary chicory root at 4% had beneficial effects on the gut health of pigs as indicated by a changed abundance of certain cecal proteins such as KRT20, SERPINB1, HSP27, ANAXA2 and ANAXA4. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection leads to severe lung damage due to pneumonia and, in more severe cases, leads to acute respiratory distress syndrome, or ARDS. This affects the viability of bronchoalveolar cells. An important role in the pathogenesis of these complications is the hyperactivation of the renin-angiotensin-aldosterone (RAA) pathway and induction of cytokine storm that occurs in an Nlrp3 inflammasome-dependent manner. To shed more light on the susceptibility of lung tissue to SARS-CoV-2 infection, we evaluated murine bronchioalveolar stem cells (BASC), alveolar type II cells (AT2), and 3D-derived organoids expression of mRNA encoding genes involved in virus entry into cells, components of RAA, and genes that comprise elements of the Nlrp3 inflammasome pathway. We noticed that all these genes are expressed by lung alveolar stem cells and organoids-derived from these cells. Interestingly, all these cells express a high level of ACE2 that, on the one hand, serves as an entry receptor for SARS-CoV-2 and, on the other, converts angiotensin II into its physiological antagonist, angiotensin 1–7 (Ang 1–7), which has been reported to have a protective role in lung damage. To shed more light on the role of Ang 1–7 on lung tissue, we exposed lung-derived BASC and AT2 cells to this mediator of RAA and noticed that it increases the proliferation of these cells. Based on this, Ang 1–7 could be employed to alleviate the damage to lung alveolar stem/progenitor cells during SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

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.Graphical Abstract: 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. [ABSTRACT FROM AUTHOR]

We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, trans-endothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs. [ABSTRACT FROM AUTHOR]

Background: Alveolar type 2 (AT2) cells and bronchioalveolar stem cells (BASC) perform critical regenerative functions in response to lung damage. Published data show that nonhematopoietic, bone marrow-derived "very small embryonic-like stem cells" (VSELs) can differentiate in vivo into surfactant protein C (SPC)-producing AT2 cells in the lung. Here, we test directly whether VSEL-derived BASC and AT2 cells function to produce differentiated progeny. Methods: using a reporter mouse in which the H2B-GFP fusion protein is driven from the murine SPC promoter, we tested whether bone marrow-derived VSELs or non-VSEL/nonhematopoietic stem cells (non-VSEL/non-HSCs) can differentiate into AT2 and BASC cells that function as progenitor cells. Immediately following bleomycin administration, WT recipient mice underwent intravenous administration of VSELs or non-VSEL/non-HSCs from SPC H2B-GFP mice. GFP+ AT2 and BASC were isolated and tested for progenitor activity using in vitro organoid assays. Results: after 21 days in vivo, we observed differentiation of VSELs but not non-VSEL/non-HSCs into phenotypic AT2 and BASC consistent with previous data in irradiated recipients. Subsequent in vitro organoid assays revealed that VSEL-derived AT2 and BASC maintained physiological potential for differentiation and self-renewal. Conclusion: these findings prove that VSELs produce functional BASC and AT2 cells, and this may open new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury. [ABSTRACT FROM AUTHOR]

Objective: Bone marrow contains a subset of stem cells that give rise to nonhematopoietic lineages. These nonhematopoietic stem cells appear heterogeneous and contain cells committed to mesenchymal and endothelial lineages, as well as more primitive multipotential cells resembling progenitors of germ cells and very small embryonic/epiblast-like stem cells (VSELs). Nonhematopoietic stem cells can be mobilized from the bone marrow in response to tissue injury, and cells with similar properties have been found in cord blood and normal adult organs. However, the relationship between bone marrow cells and these adult organ stem cells is still unclear. The differentiation potential of some adult stem cells is organ-restricted, but other populations appear to retain multipotential capacity. Materials and Methods: A population of small Sca-1+, lineage-negative (Lin−), CD45− cells resembling VSELs were isolated from neonatal mouse retina by cell sorting. Differentiation of the cells in culture was achieved by exposure to embryonic stem cell differentiation protocols. Results: VSEL-like cells comprise 1.5% of the neonatal mouse retina. They remain quiescent during retinal differentiation, and thus they do not contribute to normal retinal development. However, they display eye cell differentiation potential in culture and they are also multipotential and can give rise to cells representative of all three embryonic layers. Conclusions: The neonatal retina is an abundant postnatal source of multipotential VSEL-like cells that can differentiate in culture into a variety of lineages. [Copyright &y& Elsevier]

The liver-derived circulating in peripheral blood and intrinsic cell-expressed complement known as complosome orchestrate the trafficking of hematopoietic stem/progenitor cells (HSPCs) both during pharmacological mobilization and homing/engraftment after transplantation. Our previous research demonstrated that C3 deficient mice are easy mobilizers, and their HSPCs engraft properly in normal mice. In contrast, C5 deficiency correlates with poor mobilization and defects in HSPCs' homing and engraftment. The trafficking of HSPCs during mobilization and homing/engraftment follows the sterile inflammation cues in the BM microenvironment caused by stress induced by pro-mobilizing drugs or myeloablative conditioning for transplantation. Therefore, to explain deficiencies in HSPC trafficking between C3-KO and C5-KO mice, we evaluated the responsiveness of C3 and C5 deficient cells to low oxidative stress. As reported, oxidative stress in BM is mediated by the activation of purinergic signaling, which is triggered by the elevated level of extracellular adenosine triphosphate (eATP) and by the activation of the complement cascade (ComC). In the current work, we noticed that BM lineage negative cells (lin - ) isolated from C3-KO mice display several mitochondrial defects reflected by an impaired ability to adapt to oxidative stress. In contrast, C5-KO-derived BM cells show a high level of adaptation to this challenge. To support this data, C3-KO BM lin - cells were highly responsive to eATP stimulation, which correlates with enhanced levels of reactive oxygen species (ROS) generation and more efficient activation of intracellular Nlrp3 inflammasome. We conclude that the enhanced sensitivity of C3-KO mice cells to oxidative stress and better activation of the Nox2-ROS-Nlrp3 inflammasome signaling axis explains the molecular level differences in trafficking between C3- and C5-deficient HSPCs., (© 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).)

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).)

Very small embryonic like stem cells (VSELs) are a dormant population of stem cells that, as proposed, are deposited during embryogenesis in various tissues, including bone marrow (BM). These cells are released under steady state conditions from their tissue locations and circulate at a low level in peripheral blood (PB). Their number increases in response to stressors as well as tissue/organ damage. This increase is evident during neonatal delivery, as delivery stress prompts enrichment of umbilical cord blood (UCB) with VSELs. These cells could be purified from BM, PB, and UCB by multiparameter sorting as a population of very small CXCR4 + Lin - CD45 - cells that express the CD34 or CD133 antigen. In this report, we evaluated a number of CD34 + Lin - CD45 - and CD133 + Lin - CD45 - UCB-derived VSELs. We also performed initial molecular characterization of both cell populations for expression of selected pluripotency markers and compared these cells at the proteomic level. We noticed that CD133 + Lin - CD45 - population is more rare and express, at a higher level, mRNA for pluripotency markers Oct-4 and Nanog as well as the stromal-derived factor-1 (SDF-1) CXCR4 receptor that regulates trafficking of these cells, however both cells population did not significantly differ in the expression of proteins assigned to main biological processes., (© 2023 International Society for Advancement of Cytometry.)

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).)

Mobilization or egress of stem cells from bone marrow (BM) into peripheral blood (PB) is an evolutionary preserved and important mechanism in an organism for self-defense and regeneration. BM-derived stem cells circulate always at steady-state conditions in PB, and their number increases during stress situations related to (a) infections, (b) tissue organ injury, (c) stress, and (d) strenuous exercise. Stem cells also show a circadian pattern of their PB circulating level with peak in early morning hours and nadir late at night. The number of circulating in PB stem cells could be pharmacologically increased after administration of some drugs such as cytokine granulocyte colony-stimulating factor (G-CSF) or small molecular antagonist of CXCR4 receptor AMD3100 (Plerixafor) that promote their egress from BM into PB and lymphatic vessels. Circulating can be isolated from PB for transplantation purposes by leukapheresis. This important homeostatic mechanism is governed by several intrinsic complementary pathways. In this chapter, we will discuss the role of purinergic signaling and extracellular nucleotides in regulating this process and review experimental strategies to study their involvement in mobilization of various types of stem cells that reside in murine BM., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

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).)

Background: Diabetic patients present an increased risk for heart failure (HF) independently of the presence of coronary artery disease (CAD) and hypertension. However, little is known about circulatory microRNA (miRNA), an important regulatory RNA in this population., Objectives: To evaluate serum miRNA profile of patients with diabetes mellitus (DM) and HF and analyze its relationship with pathophysiological pathways involved., Material and Methods: The accumulation of 179 miRNAs was measured in serum of diabetic patients with HF and compared to the same measurements in healthy control subjects. The miRNAs were assayed using quantitative polymerase chain reaction (qPCR) on the Serum/Plasma Focus microRNA PCR panel (Qiagen) with LightCycler® 96 Real-Time PCR System (Roche). A pairwise comparison of mean relative miRNA accumulation levels was performed to establish those miRNAs that are differently expressed in patients with: 1) HF; 2) HF and chronic coronary syndrome (HF-CAD); and 3) HF without chronic coronary syndrome (HF-nonCAD) compared to healthy controls. To gain insight into these functions of miRNAs, we applied Gene Ontology (GO) enrichment analysis of Biological Processes and Molecular Functions of their predicted targets., Results: The pairwise comparison revealed that 12 miRNAs were significantly downregulated in HF-CAD patients compared to controls, whereas 4 miRNAs were considerably deregulated in HF-nonCAD patients, with miRNA-15b-5p being downregulated in both groups. The GO analysis revealed that differentially accumulated targets of miRNAs include genes involved in potassium channel function, MAPK kinase activity and DNA transcription regulation, with similar alterations observed in the whole HF group and HF-CAD subgroup as well as a response to stress and apoptosis (in HF group), and genes involved in the development (in HF-CAD group). No oriented specialization of deregulated miRNA targets was observed in the HF-nonCAD subgroup., Conclusion: We observed a significant downregulation of 13 miRNAs in diabetic HF patients, which was not reported previously either in HF or diabetic patients. Downregulated miRNAs regulate angiogenesis and apoptosis.

We postulated that mobilization, homing, and engraftment of hematopoietic stem/progenitor cells (HSCPs) is facilitated by a state of sterile inflammation induced in bone marrow (BM) after administration of pro-mobilizing drugs or in response to pre-transplant myeloablative conditioning. An important role in this phenomenon plays purinergic signaling that by the release of extracellular adenosine triphosphate (eATP) activates in HSPCs and in cells in the hematopoietic microenvironment an intracellular pattern recognition receptor (PPR) known as Nlrp3 inflammasome. We reported recently that its deficiency results in defective trafficking of HSPCs. Moreover, it is known that eATP after release into extracellular space is processed by cell surface expressed ectonucleotidases CD39 and CD73 to extracellular adenosine (eAdo) that in contrast to eATP shows an anti-inflammatory effect. Based on data that the state of sterile inflammation promotes trafficking of HSPCs, and since eAdo is endowed with anti-inflammatory properties we become interested in how eAdo will affect the mobilization, homing, and engraftment of HSPCs and which of eAdo receptors are involved in these processes. As expected, eAdo impaired HSPCs trafficking and this occurred in autocrine- and paracrine-dependent manner by direct stimulation of these cells or by affecting cells in the BM microenvironment. We report herein for the first time that this defect is mediated by activation of the A 2B receptor and a specific inhibitor of this receptor improves eAdo-aggravated trafficking of HSPCs. To explain this at the molecular level eAdo-A 2B receptor interaction upregulates in HSPCs in NF-kB-, NRF2- and cAMP-dependent manner heme oxygenase-1 (HO-1), that is Nlrp3 inflammasome inhibitor. This corroborated with our analysis of proteomics signature in murine HSPCs exposed to eAdo that revealed that A 2B inhibition promotes cell migration and proliferation. Based on this we postulate that blockage of A 2B receptor may accelerate the mobilization of HSPCs as well as their hematopoietic reconstitution and this approach could be potentially considered in the future to be tested in the clinic., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

Introduction: Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking., Material and Methods: We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking., Results: We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499., Conclusions: This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs.

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., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aging is an inevitable consequence of life, and all multicellular organisms undergo a decline in tissue and organ functions as they age. Several well-known risk factors, such as obesity, diabetes, and lack of physical activity that lead to the cardiovascular system, decline and impede the function of vital organs, ultimately limit overall life span. Over recent years, aging research has experienced an unparalleled growth, particularly with the discovery and recognition of genetic pathways and biochemical processes that control to some extent the rate of aging.In this chapter, we focus on several aspects of stem cell biology and aging, beginning with major cellular hallmarks of aging, endocrine regulation of aging and its impact on stem cell compartment, and mechanisms of increased longevity. We then discuss the role of epigenetic modifications associated with aging and provide an overview on a most recent search of antiaging modalities.

Recently, our group purified a rare population of primitive Sca1(+)/Lin(-)/CD45(-) cells from murine bone marrow by employing multiparameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). In order to better characterize VSELs, we focused on their morphological parameters (e.g. diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. To examine the morphological features of VSELs, we employed a multi-dimensional approach, including (i) traditional flow cytometry, (ii) a novel approach, which is ImageStream (IS) cytometry and (iii) confocal microscopy. We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than haematopoetic stem cells (HSC) (3.63 +/- 0.09 versus 6.54 +/-0.17 microm in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells in BM were detected by different investigators using different experimental strategies and hence were assigned different names (e.g., mesenchymal stem cells, multipotent adult progenitor cells, or marrow-isolated adult multilineage inducible cells). However, the search still continues for true pluripotent stem cells in adult BM, which would fulfill the required criteria (e.g. complementation of blastocyst development). Recently our group has identified in BM a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells and are found during early embryogenesis in the epiblast of the cylinder-stage embryo.

Aims: The aim of the study was to assess the correlation between the number of CD34(+), CD117(+), c-met(+), CXCR4(+) stem cells mobilized into peripheral blood, left ventricular ejection fraction (LVEF), NT-proBNP levels, and myocardial necrosis markers in patients with acute myocardial infarction (AMI)., Methods and Results: 43 patients with STEMI were enrolled. Stem cells number was measured using flow-cytometer and concentrations of NT-proBNP, SDF-1, G-CSF, VEGF, IL-6, and HGF were measured using ELISA kits. The number of stem cells mobilized early (<12 h) in AMI was significantly, positively correlated with LVEF: r=0.49 (P=0.0012) for CD34(+) cells, r=0.48 (P=0.0018) for CXCR4(+) cells, r=0.45 (P=0.0043) for CD117(+) cells, and r=0.41 (P=0.01) for c-met(+) cells and negatively correlated with NT-proBNP levels on admission r=-0.35 (P=0.024) for CD34(+) cells, r=-0.42 (P=0.007) for CXCR4(+) cells, r=-0.33 (P=0.04). In patients with LVEF 40%. The number of CXCR4(+) cells on admission and after 24 h was negatively correlated with respective cardiac Troponin I levels (r=-0.37; P=0.029 and r=-0.45, P=0.02) and maximum activity of CK-MB (r=-0.37; P=0.021). No significant correlations between levels of haematopoietic cytokines and LVEF were found., Conclusion: The mobilization of CD34(+), CD117(+), CXCR4(+), c-met(+) stem cells into peripheral blood early in STEMI is positively correlated with LVEF and negatively correlated with NT-proBNP levels and myocardial necrosis markers.

Background: Adult stem cells can contribute to myocardial regeneration after ischemic injury. Bone marrow and skeletal muscles contain a population of CXCR4+ cells expressing genes specific for muscle progenitor cells that can be mobilized into the peripheral blood. The aims of the study were (1) to confirm the presence of early tissue-committed cells expressing cardiac, muscle, and endothelial markers in populations of mononuclear cells in peripheral blood and (2) to assess the dynamics and magnitude of the mobilization of CD34+, CD117+, CXCR4+, c-met+, CD34/CD117+, and CD34/CXCR4+ stem cells into peripheral blood in relation to inflammatory and hematopoietic cytokines in patients with ST-segment-elevation acute myocardial infarction (STEMI)., Methods and Results: Fifty-six patients with STEMI (<12 hours), 39 with stable angina, and 20 healthy control subjects were enrolled. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of tissue-specific markers. The number of the cells was assessed by use of a flow cytometer on admission, after 24 hours, and after 7 days. RT-PCR revealed increased expression of mRNA (up to 3.5-fold increase) for specific cardiac (GATA4, MEF2C, Nkx2.5/Csx), muscle (Myf5, Myogenin, MyoD), and endothelial (VE-cadherin, von Willebrand factor) markers in peripheral blood mononuclear cells. The number of CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells in peripheral blood was significantly higher in STEMI patients than in stable angina and healthy subjects, peaking on admission, without further significant increase after 24 hours and 7 days., Conclusions: The study demonstrates in the setting of STEMI a marked mobilization of mononuclear cells expressing specific cardiac, muscle, and endothelial markers as well as CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells and shows that stromal cell-derived factor-1 is an important factor influencing the mobilization.