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Recent evidence indicates that extracellular adenosine triphosphate (eATP), as a major mediator of purinergic signaling, plays an important role in regulating the mobilization and homing of hematopoietic stem progenitor cells (HSPCs). In our previous work we demonstrated that eATP activates the P2X7 ion channel receptor in HSPCs and that its deficiency impairs stem cell trafficking. To learn more about the role of the P2X purinergic receptor family in hematopoiesis, we phenotyped murine and human HSPCs with respect to the seven P2X receptors and observed that, these cells also highly express P2X4 receptors, which shows ~50% sequence similarity to P2X7 subtypes, but that P2X4 cells are more sensitive to eATP and signal much more rapidly. Using the selective P2X4 receptor antagonist PSB12054 as well as P2X4-KO mice, we found that the P2X4 receptor, similar to P2X7 receptor, promotes trafficking of HSPCs in that its deficiency leads to impaired chemotaxis of HSPCs in response to a stromal-derived factor 1 (SDF-1) gradient, less effective pharmacological mobilization, and defective homing and engraftment of HSPCs after transplantation into myeloablated hosts. This correlated with a decrease in SDF-1 expression in the BM microenvironment. Overall, our results confirm the proposed cooperative dependence of both receptors in response to eATP signaling. In G-CSF-induced mobilization, a lack of one receptor is not compensated by the presence of the other one, which supports their mutual dependence in regulating HSPC trafficking. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) as an active hematopoietic organ is highly sensitive to changes in body microenvironments and responds to external physical stimuli from the surrounding environment. In particular, BM tissue responds to several cues related to infections, strenuous exercise, tissue/organ damage, circadian rhythms, and physical challenges such as irradiation. These multiple stimuli affect BM cells to a large degree through a coordinated response of the innate immunity network as an important guardian for maintaining homeostasis of the body. Innate immunity mediates these processes by engaging extracellular nucleotides, in addition to certain peptide-based factors, and bioactive phosphosphingolipids. Recent evidence demonstrates a significant role of extracellular ATP (eATP) secreted from cell in BM that is a major mediator of purinergic signaling being involved in the trafficking of hematopoietic stem/progenitor cells (HSPCs) during 1) pharmacological mobilization in their egress from the BM into peripheral blood (PB), and 2) in the reverse process of HSPCs homing and subsequent engraftment into BM after hematopoietic transplantation. While eATP stimulates positively these processes, its metabolite extracellular adenosine (eAdo) lays an opposite role. More importantly, the biological effects of eATP depend on activation in P2X7 and P2X4 receptor-dependent manner of intracellular pattern recognition receptor Nlrp3 inflammasome. Elucidation of these mechanisms will allow development of more efficient stem cell mobilization protocols to harvest the required number of HSPCs for transplantation and to accelerate hematopoietic reconstitution in patients undergoing hematopoietic transplantations. [ABSTRACT FROM AUTHOR]

The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results. [ABSTRACT FROM AUTHOR]

The aim of this article is to provide a brief review about the ImageStream system a novel tool for multiparameter cell analysis in flow. The instrument integrates the features of flow cytometry and fluorescence microscopy combined with a modern methodology for image analysis. Similar to flow cytometry, ImageStream allows analysis of a large number of cells based on their fluorescence features and provides statistical analysis of these features. Additionally, ImageStream allows detailed morphometric cellular analysis based on acquired cellular images integrating various morphometric and photometric features of the examined cells. Simply stated, ImageStream system is an advanced flow cytometer acquiring both integrated fluorescence signals as well as high quality fluorescence images and allowing muliparameter analysis. The innovative features of the instrument offer new analytical capabilities and allow for a multitude of possible applications beyond the current means of flow cytometry. While this article summarizes basic information about the features of ImageStream and its applications based on the available literature and it also describes our own experience. [ABSTRACT FROM AUTHOR]

Summary. In this study, quiescent bone marrow-derived CD34[sup +] erythroid burst-forming units (BFU-E) were found to be resistant to the inhibitory effects of tumour necrosis factor (TNF)-α and -β as well as interferon (IFN)-α, -β and -γ, in contrast to those stimulated by a combination of erytrhropoietin (Epo) plus kit ligand (KL). Unexpectedly, we found that TNF-α also inhibited the apoptosis of quiescent normal human CD34[sup +] BFU-E cells. Accordingly, TNF-α added to CD34[sup +] cells cultured for 2 d in serum-free medium protected clonogeneic BFU-E from undergoing serum deprivation-mediated apoptosis. Furthermore, the prosurvival effect of TNF-α in quiescent CD34[sup +] cells was consistent with its ability to induce phosphorylation of mitogen-activated protein kinase (MAPK) p42/44. However, when added to CD34[sup +] cells that were stimulated by Epo + KL, TNF-α induced apoptosis and inhibited proliferation of BFU-E. To explain this intriguing differential sensitivity between unstimulated CD34[sup +] cells versus those stimulated by Epo + KL, we examined the expression of apoptosis-regulating genes (FLIP , BCL-2, BCL-XL , BAD and BAX ) in these cells. Of all the genes tested, FLIP became rapidly downregulated in CD34[sup +] cells 24 h after stimulation with Epo + KL, suggesting that it may protect quiescent CD34[sup +] BFU-E progenitors residing in the bone marrow from the inhibitory effects of inflammatory cytokines. Thus, we hypothesize that cycling cells may become more sensitive to proapoptotic stimuli (e.g. chemotherapy, inhibitory cytokines) than quiescent ones because of the downregulation of protective FLIP. [ABSTRACT FROM AUTHOR]

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.Graphical Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]