The Different Responsiveness of C3- and C5-deficient Murine BM Cells to Oxidative Stress Explains Why C3 Deficiency, in Contrast to C5 Deficiency, Correlates with Better Pharmacological Mobilization and Engraftment of Hematopoietic Cells.

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.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

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.

Murine and Human-Purified very Small Embryonic-like Stem Cells (VSELs) Express Purinergic Receptors and Migrate to Extracellular ATP Gradient.

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.

Hematopoiesis Revolves Around the Primordial Evolutional Rhythm of Purinergic Signaling and Innate Immunity - A Journey to the Developmental Roots.

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.

Leukemogenesis occurs in a microenvironment enriched by extracellular microvesicles/exosomes: recent discoveries and questions to be answered.

In single-cell organisms, extracellular microvesicles (ExMVs) were one of the first cell-cell communication platforms that emerged very early during evolution. Multicellular organisms subsequently adapted this mechanism. Evidence indicates that all types of cells secrete these small circular structures surrounded by a lipid membrane that may be encrusted by ligands and receptors interacting with target cells and harboring inside a cargo comprising RNA species, proteins, bioactive lipids, signaling nucleotides, and even entire organelles "hijacked" from the cells of origin. ExMVs are secreted by normal cells and at higher levels by malignant cells, and there are some differences in their cargo. On the one hand, ExMVs secreted from malignant cells interact with cells in the microenvironment, and in return, they are exposed by a "two-way mechanism" to ExMVs secreted by non-leukemic cells. Therefore, leukemogenesis occurs and progresses in ExMVs enriched microenvironments, and this biological fact has pathologic, diagnostic, and therapeutic implications. We are still trying to decipher this intriguing cell-cell communication language better. We will present a current point of view on this topic and review some selected most recent discoveries and papers.

Mesenchymal Stem Cell-Macrophage Crosstalk Provides Specific Exosomal Cargo to Direct Immune Response Licensing of Macrophages during Inflammatory Responses.

Neurodegenerative diseases (NDDs) continue to be a significant healthcare problem. The economic and social implications of NDDs increase with longevity. NDDs are linked to neuroinflammation and activated microglia and astrocytes play a central role. There is a growing interest for stem cell-based therapy to deliver genes, and for tissue regeneration. The promise of mesenchymal stem cells (MSC) is based on their availability as off-the-shelf source, and ease of expanding from discarded tissues. We tested the hypothesis that MSC have a major role of resetting activated microglial cells. We modeled microglial cell lines by using U937 cell-derived M1 and M2 macrophages. We studied macrophage types, alone, or in a non-contact culture with MSCs. MSCs induced significant release of exosomes from both types of macrophages, but significantly more of the M1 type. RNA sequencing showed enhanced gene expression within the exosomes with the major changes linked to the inflammatory response, including cytokines and the purinergic receptors. Computational analyses of the transcripts supported the expected effect of MSCs in suppressing the inflammatory response of M1 macrophages. The inflammatory cargo of M1 macrophage-derived exosomes revealed involvement of cytokines and purinergic receptors. At the same time, the exosomes from MSC-M2 macrophages were able to reset the classical M2 macrophages to more balanced inflammation. Interestingly, we excluded transfer of purinergic receptor transcripts from the co-cultured MSCs by analyzing these cells for the identified purinergic receptors. Since exosomes are intercellular communicators, these findings provide insights into how MSCs may modulate tissue regeneration and neuroinflammation.

Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) by Downregulating Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) Activity.

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.

The "Mystery Cell Population" Residing in Murine Bone Marrow - A Missing Link Between Very Small Embryonic Like Stem Cells and Hematopoietic Stem Cells?

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.

Molecular analysis and comparison of CD34+ and CD133+ very small embryonic-like stem cells purified from umbilical cord blood.

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.

Hematopoietic stem cells on the crossroad between purinergic signaling and innate immunity.

Hematopoiesis is regulated by several mediators such as peptide-based growth factors, cytokines, and chemokines, whose biological effects have been studied for many years. However, several other mediators have been identified recently that affect the fate of hematopoietic stem/progenitor cells (HSPC) as well as non-hematopoietic cells in the bone marrow microenvironment. These new mediators comprise members of purinergic signaling pathways and are active mediators of the soluble arm of innate immunity, the complement cascade (ComC). In this review, we will discuss the coordinated effects of these pathways in regulating the biology of HSPC. Importantly, both purinergic signaling and the ComC are activated in stress situations and interact with specific receptors expressed on HSPC. Evidence has accumulated indicating that some of the purinergic as well as ComC receptors could also be activated intracellularly by intrinsically expressed ligands. To support this recent evidence, our work indicates that the major mediator of purinergic signaling, adenosine triphosphate, and the cleavage product of the fifth component of the ComC (C5), C5a anaphylatoxin, can activate their corresponding receptors expressed on the outer mitochondrial membrane in an autocrine manner. We will also discuss recent evidence that these responses, mediated by purinergic signaling and the ComC network, are coordinated by activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 - reactive oxygen species - NLR family pyrin domain containing 3 (NLRP3) inflammasome (Nox2-ROS-NLRP3) axis.

External Liver-Derived Complement and Intrinsic Present in Hematopoietic Stem/Progenitor Cells Complosome Modulate Cell Metabolism and Response to Stress.

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.

Complement Activation Products in Patients with Chronic Schizophrenia.

Evidence suggests a role of the immune system in the pathogenesis of a number of mental conditions, including schizophrenia (SCH). In terms of physiology, aside from its crucial protective function, the complement cascade (CC) is a critical element of the regeneration processes, including neurogenesis. Few studies have attempted to define the function of the CC components in SCH. To shed more light on this topic, we compared the levels of complement activation products (CAP) (C3a, C5a and C5b-9) in the peripheral blood of 62 patients with chronic SCH and disease duration of ≥ 10 years with 25 healthy controls matched for age, sex, BMI and smoking status. Concentrations of all the investigated CAP were elevated in SCH patients. However, after controlling for potential confounding factors, significant correlations were observed between SCH and C3a (M = 724.98 ng/mL) and C5a (M = 6.06 ng/mL) levels. In addition, multivariate logistic regression showed that C3a and C5b-9 were significant predictors of SCH. There were no significant correlations between any CAP and SCH symptom severity or general psychopathology in SCH patients. However, two significant links emerged between C3a and C5b-9 and global functioning. Increased levels of both complement activation products in the patient group as compared to healthy controls raise questions concerning the role of the CC in the etiology of SCH and further demonstrate dysregulation of the immune system in SCH patients.

The Nox2-ROS-Nlrp3 Inflammasome Signaling Stimulates in the Hematopoietic Stem/Progenitor Cells Lipogenesis to Facilitate Membrane Lipid Raft Formation.

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.

Combined Transplantation of Human MSCs and ECFCs Improves Cardiac Function and Decrease Cardiomyocyte Apoptosis After Acute Myocardial Infarction.

BACKGROUND: Ischemic heart disease, often caused by an acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide. Despite significant advances in medical and procedural therapies, millions of AMI patients progress to develop heart failure every year. METHODS: Here, we examine the combination therapy of human mesenchymal stromal cells (MSCs) and endothelial colony-forming cells (ECFCs) to reduce the early ischemic damage (MSCs) and enhance angiogenesis (ECFCs) in a pre-clinical model of acute myocardial infarction. NOD/SCID mice were subjected to AMI followed by transplantation of MSCs and ECFCs either alone or in combination. Cardiomyocyte apoptosis and cardiac functional recovery were assessed in short- and long-term follow-up studies. RESULTS: At 1 day after AMI, MSC- and ECFC-treated animals demonstrated significantly lower cardiomyocyte apoptosis compared to vehicle-treated animals. This phenomenon was associated with a significant reduction in infarct size, cardiac fibrosis, and improvement in functional cardiac recovery 4 weeks after AMI. CONCLUSIONS: The use of ECFCs, MSCs, and the combination of both cell types reduce cardiomyocyte apoptosis, scar size, and adverse cardiac remodeling, compared to vehicle, in a pre-clinical model of AMI. These results support the use of this combined cell therapy approach in future human studies during the acute phase of ischemic cardiac injury.

Proteomic Analysis of Murine Bone Marrow Very Small Embryonic-like Stem Cells at Steady-State Conditions and after In Vivo Stimulation by Nicotinamide and Follicle-Stimulating Factor Reflects their Germ-Lineage Origin and Multi Germ Layer Differentiation Potential.

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.

Purinergic Signaling and Its Role in Mobilization of Bone Marrow Stem Cells.

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.