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.

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.

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.

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.

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.

Novel evidence that the P2X1 purinergic receptor-Nlrp3 inflammasome axis orchestrates optimal trafficking of hematopoietic stem progenitors cells.

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.

Extracellular Adenosine (eAdo) - A2B Receptor Axis Inhibits in Nlrp3 Inflammasome-dependent Manner Trafficking of Hematopoietic Stem/progenitor Cells.

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.

Myeloablative Conditioning for Transplantation Induces State of Sterile Inflammation in the Bone Marrow: Implications for Optimizing Homing and Engraftment of Hematopoietic Stem Cells.

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.

Novel Evidence That Alternative Pathway of Complement Cascade Activation is Required for Optimal Homing and Engraftment of Hematopoietic Stem/progenitor Cells.

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.

The P2X4 purinergic receptor has emerged as a potent regulator of hematopoietic stem/progenitor cell mobilization and homing-a novel view of P2X4 and P2X7 receptor interaction in orchestrating stem cell trafficking.

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.

Danger-associated molecular pattern molecules take unexpectedly a central stage in Nlrp3 inflammasome-caspase-1-mediated trafficking of hematopoietic stem/progenitor cells.

Like their homing after transplantation to bone marrow (BM), the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not fully understood, and several overlapping pathways are involved. Several years ago our group proposed that sterile inflammation in the BM microenvironment induced by pro-mobilizing agents is a driving force in this process. In favor of our proposal, both complement cascade (ComC)-deficient and Nlrp3 inflammasome-deficient mice are poor G-CSF and AMD3100 mobilizers. It is also known that the Nlrp3 inflammasome mediates its effects by activating caspase-1, which is responsible for proteolytic activation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) and their release from cells along with several danger-associated molecular pattern molecules (DAMPs). We observed in the past that IL-1ß and IL-18 independently promote mobilization of HSPCs. In the current work we demonstrated that caspase-1-KO mice are poor mobilizers, and, to our surprise, administration of IL-1ß or IL-18, as in the case of Nlrp3-KO animals, does not correct this defect. Moreover, neither Caspase-1-KO nor Nlrp3-KO mice properly activated the ComC to execute the mobilization process. Interestingly, mobilization in these animals and activation of the ComC were both restored after injection of the DAMP cocktail eATP+HGMB1+S100A9, the components of which are normally released from cells in an Nlrp3 inflammasome-caspase-1-dependent manner. In addition, we report that caspase-1-deficient HSPCs show a decrease in migration in response to BM homing factors and engraft more poorly after transplantation. These results for the first time identify caspase-1 as an orchestrator of HSPC trafficking.

Innate Immunity Communicates Using the Language of Extracellular Microvesicles.

The innate immunity system and extracellular microvesicles (ExMVs) both emerged early in the evolution of life, which is why its innate immunity cellular arm and its soluble-component arm learned, understood, and adapted to the "language" of ExMVs. This was most likely the first language of cell-cell communication during evolution, which existed before more specific intercellular crosstalk involving specific ligands and receptors emerged. ExMVs are involved in several processes in the body, including immune and coagulation responses, which are part of inflammation. In this review we will briefly highlight what is known about how ExMVs regulate the function of the cellular arm of innate immunity, including macrophages, monocytes, granulocytes, natural killer cells, and dendritic cells, and affect the soluble components of this system, which consists of the complement cascade (ComC) and soluble, circulating, pattern-recognition receptors (collectins, ficolins, and pentaxrins). These effects are direct, due to the fact that ExMVs affect the biological functions of innate immunity cells and may directly interact with soluble components of this system. Moreover, by activating coagulation proteases, ExMVs may also indirectly activate the ComC. In this review, we will use the term "extracellular microvesicles" (ExMVs) to refer to these small, spheroidal blebs of different sizes, which are surrounded by a membrane lipid layer. We will focus on the role of both ExMVs released during cell-surface membrane budding and smaller ExMVs, known as exosomes, which are derived from the budding of the endosomal membrane compartment. Finally, we will provide a brief update on the potential therapeutic applications of ExMVs, with a special emphasis on innate immunity.

Heme Oxygenase 1 (HO-1) as an Inhibitor of Trafficking of Normal and Malignant Hematopoietic Stem Cells - Clinical and Translational Implications.

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.

Extracellular microvesicles/exosomes: discovery, disbelief, acceptance, and the future?

There are concepts in science that need time to overcome initial disbelief before finally arriving at the moment when they are embraced by the research community. One of these concepts is the biological meaning of the small, spheroidal vesicles released from cells, which are described in the literature as microparticles, microvesicles, or exosomes. In the beginning, this research was difficult, as it was hard to distinguish these small vesicles from cell debris or apoptotic bodies. However, they may represent the first language of cell-cell communication, which existed before a more specific intercellular cross-talk between ligands and receptors emerged during evolution. In this review article, we will use the term "extracellular microvesicles" (ExMVs) to refer to these small spheroidal blebs of different sizes surrounded by a lipid layer of membrane. We have accepted an invitation from the Editor-in-Chief to write this review in observance of the 20th anniversary of the 2001 ASH Meeting when our team demonstrated that, by horizontal transfer of several bioactive molecules, including mRNA species and proteins, ExMVs harvested from embryonic stem cells could modify hematopoietic stem/progenitor cells and expand them ex vivo. Interestingly, the result that moved ExMV research forward was published first in 2005 in Leukemia, having been previously rejected by other major scientific journals out of simple disbelief. Therefore, the best judge of a new concept is the passage of time, although the speed of its adoption is aided by perseverance and confidence in one's own data. In this perspective article, we will provide a brief update on the current status of, hopes for, and likely future of ExMV research as well as therapeutic and diagnostic applications, with a special emphasis on hematopoiesis.