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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.

The scientific community faces an unexpected and urgent challenge related to the SARS-CoV-2 pandemic and is investigating the role of receptors involved in entry of this virus into cells as well as pathomechanisms leading to a cytokine “storm,” which in many cases ends in severe acute respiratory syndrome, fulminant myocarditis and kidney injury. An important question is if it may also damage hematopoietic stem progenitor cells?

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.

Recent investigations indicate that hematopoiesis is coregulated by innate immunity signals and by pathways characteristic of the activation of innate immunity cells that also operate in normal hematopoietic stem progenitor cells (HSPCs). This should not be surprising because of the common developmental origin of these cells from a hemato/lymphopoietic stem cell. An important integrating factor is the Nlrp3 inflammasome, which has emerged as a major sensor of changes in body microenvironments, cell activation, and cell metabolic activity. It is currently the best-studied member of the inflammasome family expressed in hematopoietic and lymphopoietic cells, including also HSPCs. It is proposed as playing a role in (i) the development and expansion of HSPCs, (ii) their release from bone marrow (BM) into peripheral blood (PB) in stress situations and during pharmacological mobilization, (iii) their homing to BM after transplantation, and (iv) their aging and the regulation of hematopoietic cell metabolism. The Nlrp3 inflammasome is also involved in certain hematological pathologies, including (i) myelodysplastic syndrome, (ii) myeloproliferative neoplasms, (iii) leukemia, and (iv) graft-versus-host disease (GvHD) after transplantation. The aim of this review is to shed more light on this intriguing intracellular protein complex that has become a “rising star” in studies focused on both normal steady-state and pathological hematopoiesis.

Hematopoietic stem/progenitor cells (HSPCs) circulate in peripheral blood (PB) under normal conditions and their number increases in response to stress, inflammation, tissue/organ injury, and may increase up to 100-fold after administration of mobilization-inducing drugs. Mounting evidence suggests that mobilizing agent-induced mobilization of HSPCs from bone marrow into PB is a result of innate immunity-mediated sterile inflammation in the bone marrow (BM) microenvironment. A critical initiating role in this process is played by tissue/organ injury-mediated or pharmacologically induced release from bone marrow-residing granulocytes and monocytes of (i) danger-associated molecular patterns (DAMPs), (ii) reactive oxygen species (ROS), and (iii) proteolytic and lipolytic enzymes. All these factors together trigger activation of the complement and coagulation cascades, both of which orchestrate egress of HSPCs into BM sinusoids and lymphatics. Recent evidence also indicates that, in addition to attenuation of the SDF-1–CXCR4 and VLA-4–VCAM-1 retention axes in the BM microenvironment and the presence of a mobilization-directing phosphosphingolipid gradient in PB, an important role in the mobilization process is played by extracellular nucleotides and purinergic signaling. In particular, a new finding by our laboratory is that, while extracellular ATP promotes mobilization of HSPCs, its derivative, adenosine, has the opposite (inhibitory) effect.

A population of CD133 + lin - CD45 - and CD34 + lin - CD45 - very small embryonic-like stem cells (VSELs) has been identified in postnatal human tissues, including bone marrow (BM), mobilized peripheral blood (mPB) and umbilical cord blood (UCB). Under appropriate conditions, VSELs in vitro and in vivo differentiate into tissue-committed stem cells for all three germ layers. Molecular analysis of adult murine BM-purified VSELs revealed that these rare cells deposited during development in adult tissues (i) express a similar transcriptome as embryonic stem cells, (ii) share several markers characteristic for epiblast and migratory primordial germ cells (PGCs), (iii) highly express a polycomb group protein enhancer of zeste drosophila homolog 2 (Ezh2) and finally (iv) display a unique pattern of imprinting at crucial paternally inherited genes that promotes their quiescence. Here, by employing single-cell RNA sequencing we demonstrate for the first time that purified from UCB human VSELs defined by expression of CD34 or CD133 antigens and lack of lineage markers, including CD45 antigen express similar molecular signature as murine BM-derived VSELs. Specifically, unsupervised clustering revealed numerous subpopulations of VSELs including ones i) annotated to germline compartments, ii) regulated by parental imprinting, iii) responding to early developmental fate decisions, iv) transcription factors involved in differentiation and development, including homeobox family of genes, and v) expressing innate immunity and purinergic signaling genes.

The insulin-like growth factor-2 (Igf2)-H19 locus encodes important paternally imprinted genes that govern normal embryonic development. While Igf-2 encodes IGF2, which is an autocrine/paracrine mitogen,  transcription of H19 gives rise to non-coding mRNA that is a precursor of several microRNAs (miRNAs) that negatively affect cell proliferation. The proper imprinting of a differentially methylated region (DMR) within this locus, with methylation of the paternal chromosome and a lack of methylation on the maternal chromosome, regulates expression of both of these genes so that Igf2 is transcribed only from the paternal chromosome and H19 only from the maternal chromosome. There is growing evidence that this 'Yin-Yang' locus regulates embryonic development. Furthermore, recent evidence indicates that erasure of imprinting (hypomethylation) of the Igf2-H19 locus on both chromosomes, which leads to downregulation of Igf2 and upregulation of H19 expression, plays an important role in regulating quiescence of pluripotent stem cells in adult organisms, and may be involved in the regulation of lifespan. In contrast, hypermethylation of this locus on both chromosomes (loss of imprinting) results in Igf2 overexpression and is observed in several malignancies. In this review, we will discuss the biological consequences of changes in Igf2-H19 expression.

Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed to chemo-resistance of cancer stem cells (CSCs). Herein we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera, when used alone or in combination with cisplatin (CIS) targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors generated by injecting human ovarian epithelial cancer cell line (A2780) with WFA and cisplatin (WFA) alone or in combination resulted in a 70 to 80% reduction in tumor growth and complete inhibition of metastasis to other organs compared to untreated controls. Histochemical and Western blot analysis of the tumors revealed that inclusion of WFA (2 mg/kg) resulted in a highly significant elimination of cells expressing CSC markers - CD44, CD24, CD34, CD117 and Oct4 and downregulation of Notch1, Hes1 and Hey1 genes. In contrast treatment of mice with CIS alone (6 mg/kg) had opposite effect on those cells. Increase in cells expressing CSC markers and Notch1 signaling pathway in tumors exposed to CIS may explain recurrence of cancer in patients treated with carboplatin and paclitaxel. Since, WFA alone or in combination with CIS eliminates putative CSCs, we conclude that WFA in combination with CIS may present more efficacious therapy for ovarian cancer.

Evidence has accumulated that murine haematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs. It has also been reported that human HSPCs, like their murine counterparts, respond to certain SexHs (e.g. androgens). However, to better address the effects of SexHs, particularly pituitary SexHs, on human haematopoiesis, we tested for expression of receptors for pituitary SexHs, including follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, oestrogens, and androgen, on HSPCs purified from human umbilical cord blood (UCB) and peripheral blood (PB). We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB‐derived, CD45− very small embryonic‐like stem cells (VSELs) become specified into CD45+ HSPCs, we also evaluated the expression of pituitary and gonadal SexH receptors on these cells. We report for the first time that human HSPCs and VSELs, like their murine counterparts, express pituitary and gonadal SexH receptors at the mRNA and protein levels. Most importantly, SexH if added to suboptimal doses of haematopoietic cytokines and growth factors enhance clonogenic growth of human HSPCs as well as directly stimulate proliferation of MSCs.

BackgroundThe complement system expressed intracellularly and known as complosome has been indicated as a trigger in the regulation of lymphocyte functioning. The expression of its genes was confirmed also in several types of human bone marrow-derived stem cells: mononuclear cells (MNCs), very small embryonic-like stem cells (VSELs), hematopoietic stem/progenitor cells (HSPCs), endothelial progenitors (EPCs) and mesenchymal stem cells (MSCs). In our previous studies, we demonstrated the expression of complosome proteins including C3, C5, C3aR, and cathepsin L in purified HSPCs. However, there is still a lack of results showing the expression of complosome system elements and other immunity-related proteins in human HSPCs at the level of single cell resolution.MethodsWe employed scRNA-seq to investigate comprehensively the expression of genes connected with immunity, in two populations of human HSPCs: CD34+Lin-CD45+ and CD133+Lin-CD45+, with the division to subpopulations. We focused on genes coding complosome elements, selected cytokines, and genes related to antigen presentation as well as related to immune regulation.ResultsWe observed the differences in the expression of several genes e.g. C3AR1 and C5AR1 between two populations of HSPCs: CD34+LinCD45+ and CD133+Lin-CD45+ resulting from their heterogeneous nature. However, in both kinds of HSPCs, we observed similar cell subpopulations expressing genes (e.g. NLRP3 and IL-1β) at the same level, which suggests the presence of cells performing similar functions connected with the activation of inflammatory processes contributing to the body's defense against infections.DiscussionTo our best knowledge, it is the first time that expression of complosome elements was studied in HSPCs at the single cell resolution with the use of single cell sequencing. Thus, our data sheds new light on complosome as a novel regulator of hematopoiesis that involves intracrine activation of the C5a-C5aR-Nlrp3 inflammasome axis.