The Nlrp3 Inflammasome Orchestrates Mobilization of Bone Marrow-Residing Stem Cells into Peripheral Blood.

Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a "sterile inflammation" in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1ß and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP-Nlrp3-ComC axis in the egress of stem cells into PB.

ATP-Nlrp3 Inflammasome-Complement Cascade Axis in Sterile Brain Inflammation in Psychiatric Patients and its Impact on Stem Cell Trafficking.

Recent evidence indicates that the occurrence of psychiatric disorders in patients is linked to a local "sterile" inflammation of brain or due to a systemic inflammation process that affects the central nervous system. This is supported by the observation that in peripheral blood of psychotic patients are detectable several mediators and markers of inflammation as well as clinical data on correlations between systemic chronic inflammatory processes and psychiatric disorders. This may explain why some reported anti-inflammatory treatment strategies have beneficial effects on ameliorating psychotic events. In this review we will present a concept that aberrant purinergic signaling and increases in extracellular level of adenosine triphosphate (ATP) in the brain parenchyma may lead to activation of Nlrp3 inflammasome in microglia cells and as a consequence microglia released danger associated molecular pattern (DAMP) proteins activate complement cascade (ComC) in mannan binding lectin (MBL) - dependent manner. Activation of ATP-Nlrp3 inflammasome-ComC axis may also orchestrate trafficking of stem cells released from bone marrow into peripheral blood observed in psychotic patients. Based on this, the ATP-Nlrp3 inflammasome-ComC axis may become a target for new therapeutic approaches, which justifies the development and clinical application of efficient anti-inflammatory treatment strategies targeting this axis in psychiatry.

[Corrigendum] Pituitary sex hormones enhance the pro­metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase­1.

After the publication of the article, the authors realize that they overlooked stating that the author Magda Kucia was the recipient of an OPUS grant (grant no. UMO­2016/21/B/NZ4/00201). Therefore, the Acknowledgements section of the paper should have read as follows (the added text is highlighted in bold): "This study was supported by NIH grants 2R01 DK074720 and R01HL112788, the Stella and Henry Endowment, and NCN Harmonia grant UMO­2014/14/M/NZ3/00475 to M.Z.R., and OPUS grant UMO­2016/21/B/NZ4/00201 to M.K." The authors regret their oversight in failing to include this information in the Acknowledgements section of their paper. [the original article was published in International Journal of Oncology 50: 317-328, 2017; DOI: 10.3892/ijo.2016.3787].

Sterile Inflammation of Brain, due to Activation of Innate Immunity, as a Culprit in Psychiatric Disorders.

Evidence has accumulated that the occurrence of psychiatric disorders is related to chronic inflammation. In support of this linkage, changes in the levels of circulating pro-inflammatory cytokines and chemokines in the peripheral blood (PB) of psychiatric patients as well as correlations between chronic inflammatory processes and psychiatric disorders have been described. Furthermore, an inflammatory process known as "sterile inflammation" when initiated directly in brain tissue may trigger the onset of psychoses. In this review, we will present the hypothesis that prolonged or chronic activation of the complement cascade (ComC) directly triggers inflammation in the brain and affects the proper function of this organ. Based on the current literature and our own work on mechanisms activating the ComC we hypothesize that inflammation in the brain is initiated by the mannan-binding lectin pathway of ComC activation. This activation is triggered by an increase in brain tissue of danger-associated molecular pattern (DAMP) mediators, including extracellular ATP and high-mobility group box 1 (HMGB1) protein, which are recognized by circulating pattern-recognition receptors, including mannan-binding lectin (MBL), that activate the ComC. On the other hand, this process is controlled by the anti-inflammatory action of heme oxygenase 1 (HO-1). In this review, we will try to connect changes in the release of DAMPs in the brain with inflammatory processes triggered by the innate immunity involving activation of the ComC as well as the inflammation-limiting effects of the anti-inflammatory HO-1 pathway. We will also discuss parallel observations that during ComC activation subsets of stem cells are mobilized into PB from bone marrow that are potentially involved in repair mechanisms.

Do Cancer Cell Lines Have Fixed or Fluctuating Stem Cell Phenotypes? - Studies with the NTera2 Cell Line.

One of the important questions when studying established cancer cell lines is whether such cells contain a subpopulation of primitive cancer stem cells that maintains the expansion of the cell line. To address this issue, we performed studies on the established human embryonal carcinoma cell line NTera2 by evaluating the potential stemness of cells sorted according to their expression of the cell surface stem cell markers CD133 and SSEA4. By performing in vitro and in vivo assays, we observed different properties of cells expressing both, one, or neither of these antigens. While sorted SSEA4+ subpopulations exhibited the greatest propensity for migration toward normal serum and the highest seeding efficiency in the lungs of immunodeficient mice, CD133-SSEA4- cells displayed high seeding efficiency to the bone marrow after injection in vivo. It is worth noting that these properties did not depend on the size of the evaluated cells. To address the question of whether cancer stem cell phenotypes in cell lines are fixed or fluctuating, we sorted single cells according to their expression of CD133 and SSEA4 antigens and observed that cells which did not express these cancer stem cell markers gave rise to cells that express these markers after expansion in vitro. Therefore, our results support the idea that within established cancer cell lines, the phenotype of the cell subpopulation expressing cancer stem cell markers is not fixed but fluctuates during cell line expansion, and cells negative for these markers may acquire their expression.

Pituitary sex hormones enhance the pro­metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase­1.

We report that human lung cancer cell lines express functional receptors for pituitary sex hormones (SexHs) and respond to stimulation by follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL). Expression of these receptors has also been confirmed in patient lung cancer samples at the mRNA level. Stimulation of human lung cancer cell lines with FSH, LH, or PRL stimulated migration and chemotaxis, and some cell lines responded by enhanced proliferation. Moreover, priming of human lung cancer cells by exposing them to pituitary SexHs resulted in enhanced seeding efficiency of injected human lung cancer cells into bone marrow, liver, and lungs in an immunodeficient mouse model. The chemotaxis of lung cancer cell lines corresponded with the activity of heme oxygenase­1 (HO­1), as stimulation of these cells by FSH, LH, and PRL downregulated its expression in a p38 MAPK­dependent manner. Moreover, while downregulation of HO­1 by the small­molecule inhibitor tin protoporphyrin (SnPP) promoted migration, upregulation of HO­1 by the small­molecule activator cobalt protoporphyrin (CoPP) showed the opposite effect. Based on this finding, we propose that pituitary SexHs play a significant role in the pathogenesis of lung cancer, particularly when the blood level of FSH increases due to gonadal dysfunction with advanced age. Finally, we propose that upregulation of HO­1 expression by a small­molecule activator may be effective in controlling SexH­induced cell migration in lung cancer.

Mobilization of Peripheral Blood Stem Cells and Changes in the Concentration of Plasma Factors Influencing their Movement in Patients with Panic Disorder.

In this paper we examined whether stem cells and factors responsible for their movement may serve as new biological markers of anxiety disorders. The study was carried out on a group of 30 patients diagnosed with panic disorder (examined before and after treatment), compared to 30 healthy individuals forming the control group. We examined the number of circulating HSCs (hematopoetic stem cells) (Lin-/CD45 +/CD34 +) and HSCs (Lin-/CD45 +/AC133 +), the number of circulating VSELs (very small embryonic-like stem cells) (Lin-/CD45-/CD34 +) and VSELs (Lin-/CD45-/AC133 +), as well as the concentration of complement components: C3a, C5a and C5b-9, SDF-1 (stromal derived factor) and S1P (sphingosine-1-phosphate). Significantly lower levels of HSCs (Lin-/CD45 +/AC133 +) have been demonstrated in the patient group compared to the control group both before and after treatment. The level of VSELs (Lin-/CD45-/CD133 +) was significantly lower in the patient group before treatment as compared to the patient group after treatment.The levels of factors responsible for stem cell movement were significantly lower in the patient group compared to the control group before and after treatment. It was concluded that the study of stem cells and factors associated with their movement can be useful in the diagnostics of panic disorder, as well as differentiating between psychotic and anxiety disorders.

Extracellular Microvesicles (ExMVs) in Cell to Cell Communication: A Role of Telocytes.

There are several mechanisms by which cells communicate with each other. Evidence accumulates that the evolutionary oldest mechanisms of cell-cell communication involves extracellular microvesicles (ExMVs). Generally, these circular membrane fragments enriched for mRNA, miRNA, proteins, and bioactive lipids are released by exocytosis from endosomal compartment or are directly formed by budding from cell surface membranes. ExMVs from endosomal compartment called exosomes are smaller in size ~100 nM as compared to larger ones released from cell membranes that are in size up to 1 µM. In this chapter we will present an emerging link between ExMVs and recently identified novel cell-cell communication network involving a new type of cell known as telocyte. Mounting evidence accumulates that telocytes mediate several of their biological effects in several organs by releasing ExMVs enriched in mRNA, miRNA, proteins, and several biological mediators to the target cells.

Vitamin D3 stimulates embryonic stem cells but inhibits migration and growth of ovarian cancer and teratocarcinoma cell lines.

BACKGROUND: Deficiency in Vitamin D3 (cholecalciferol) may predispose to some malignancies, including gonadal tumors and in experimental models vitamin D3 has been proven to inhibit the growth of cancer cells. To learn more about the potential role of vitamin D3 in cancerogenesis, we evaluated the expression and functionality of the vitamin D receptor (VDR) and its role in metastasis of ovarian cancer cells and of murine and human teratocarcinoma cell lines. METHODS: In our studies we employed murine embrynic stem cells (ESD3), murine (P19) and human (NTERA-2) teratocarcimona cells lines, human ovarian cancer cells (A2780) as well as purified murine and human purified very small embryonic like stem cells (VSELs). We evaluated expression of Vitamin D3 receptor (VDR) in these cells as well as effect of vitamin D3 exposure on cell proliferation and migration. RESULTS: We here provide also more evidence for the role of vitamin D3 in germline-derived malignancies, and this evidence supports the proposal that vitamin D3 treatment inhibits growth and metastatic potential of several germline-derived malignancies. We also found that the ESD3 murine immortalized embryonic stem cell line and normal, pluripotent, germline-marker-positive very small embryonic-like stem cells (VSELs) isolated from adult tissues are stimulated by vitamin D3, which suggests that vitamin D3 affects the earliest stages of embryogenesis. CONCLUSIONS: We found that however all normal and malignant germ-line derived cells express functional VDR, Vitamin D3 differently affects their proliferation and migration. We postulate that while Vitamin D3 as anticancer drug inhibits proliferation of malignant cells, it may protect normal stem cells that play an important role in development and tissue/organ regeneration.

Hematopoietic stem/progenitor cells express several functional sex hormone receptors-novel evidence for a potential developmental link between hematopoiesis and primordial germ cells.

Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45(-) very small embryonic-like stem cells (VSELs) may become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy.

An emerging question about putative cancer stem cells in established cell lines-are they true stem cells or a fluctuating cell phenotype?

It has been proposed that established cell lines contain populations of cancer stem cells (CSCs), which are responsible for expansion of these cell lines and their metastatic potential. To address this issue better, we employed a human ovarian cancer cell line, A2780, and sorted cells according to the postulated highly mestatatic cancer stem cell phenotype, CD24+CD44-, and the less-metastatic CD24-CD44+ and CD24-CD44- phenotypes. These cells were employed in chemotaxis assays in vitro to migrate in response to conditioned media harvested from bone marrow or liver cells damaged by irradiation and in in vivo assays to grow tumors after injection into immunodeficient mice. We also sorted single cells expressing all three phenotypes by FACS and expanded them to grow clones. We found that the CD24+CD44- cells are a highly migratory population compared with CD24-CD44+ and CD24-CD44- cells and were seeded in higher numbers in murine bone marrow and liver after intravenous injection. Most importantly, we observed that singly sorted cells efficiently expanded ex vivo into cell populations that represented all phenotypes of the parental cell line. Thus, our data indicate that cells expressing a certain set of markers, e.g., CD24, have at any given moment a higher potential to migrate and metastasize. However, cells that are CD24-negative, if expanded from a singly sorted cell, may give rise to cells containing all of the markers, including CD24. Based on this finding, we propose that the CSC phenotype in cell lines fluctuates with cell expansion.

Identification of heme oxygenase 1 (HO-1) as a novel negative regulator of mobilization of hematopoietic stem/progenitor cells.

Activation of complement cascade (ComC) play and important role in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). While there are vast experimental data on the mechanisms and factors that induce or promote mobilization of HSPCs, there is relatively less data on negative regulators of this process. We demonstrate for the first time that heme oxygenase-1 (HO-1) that has a well-documented anti-inflammatory potential plays an important and heretofore unrecognized role in retention of HSPCs in BM niches by i) modulating negatively activation of mobilization promoting ComC, ii) maintaining stromal derived factor-1 (SDF-1) level in the BM microenvironment and iii) attenuating chemotactic responsiveness of HSPCs to SDF-1 and sphingosine-1 phosphate (S1P) gradients in PB. Furthermore, our data showing a positive mobilizing effect by a non-toxic small-molecule inhibitor of HO-1 (SnPP) suggest that blockade of HO-1 would be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate better the molecular mechanisms responsible for the potential effect of HO-1 in homing of HSPCs after transplantation.

Positive effects of prolonged caloric restriction on the population of very small embryonic-like stem cells - hematopoietic and ovarian implications.

BACKGROUND: Low calorie intake, or calorie restriction (CR) without malnutrition, has been demonstrated in several animal species, including mice, to increase both median and maximum lifespan as well as delay reproductive senescence. Our previous work demonstrated a positive correlation between life span and the number of very small embryonic-like stem cells (VSELs) in long living Laron dwarf mice. These animals have very low levels of circulating insulin-like growth factor 1 (IGF-1) in peripheral blood (PB), maintain higher numbers of hematopoietic stem cells (HSPCs) in bone marrow (BM), and display prolonged fecundity compared with wild type littermates. Since CR lowers the level of IGF-1 in PB, we become interested in the effect of CR on the number of VSELs and HSPCs in BM as well as on the morphology of ovaries and testes. METHODS: In our studies four-week-old female and male mice were subjected to CR by employing an alternate-day ad libitum feeding diet for a period of 9 months. RESULTS: We observed that mice on CR had a higher number of BM-residing VSELs than control mice fed ad libitum. These changes correlated with higher numbers of HSPCs in BM, spleen, and peripheral blood (PB) as well as with an increase in the number of primordial and primary follicles in ovaries. At the same time, however, no changes were observed in the testes of mice under CR. CONCLUSION: We conclude that CR positively affects the pool of VSELs in adult tissues and explains the positive effect of CR on longevity.

New advances in stem cell research: practical implications for regenerative medicine.

Regenerative medicine is searching for stem cells that can be safely and efficiently employed for regeneration of damaged solid organs (e.g., the heart, brain, or liver). Ideal for this purpose would be pluripotent stem cells, which, according to their definition, have broad potential to differentiate into all types of adult cells. For almost 20 years, there have been unsuccessful attempts to harness controversial embryonic stem cells (ESCs) isolated from embryos. Induced pluripotent stem cells (iPSCs), generated by genetic modification of adult somatic cells, are a more promising source. However, both iPSC and ESCs are associated with a risk of teratoma formation. At the same time, various types of more­differentiated adult stem and progenitor cells derived from the bone marrow, umbilical cord blood, mobilized peripheral blood, or fat tissue are being employed in clinical trials to regenerate damaged solid organs. However, for most of these cells, there is a lack of convincing documentation for successful regeneration of the treated organs. Beneficial effects of those cells might be explained by paracrine effects of growth factors, cytokines, chemokines, bioactive lipids, and extracellular microvesicles, which are released from the cells and have trophic, antiapoptotic, and angiopoietic effects. Nevertheless, there is evidence that adult tissues harbor a promising population of very rare dormant stem cells with broad differentiation potential. In this review, we will discuss various potential sources of stem cells for regenerative medicine and the mechanisms that explain some of their beneficial effects as well as highlight the results of the first clinical trials.  

Novel evidence for enhanced stem cell trafficking in antipsychotic-naïve subjects during their first psychotic episode.

In this study, we tested the novel hypothesis that stem cells and those factors that modulate their trafficking may be biological markers for acute psychosis. Twenty-eight subjects during their first nonaffective psychotic episode were investigated before and after antipsychotic treatment and were compared with 35 healthy controls (CG); the psychotic group (PG) was divided into "schizophrenic" (SG) and "non-schizophrenic" (NG) subgroups. We examined the number of circulating Lin(-)/CD45(-)/CD34(+) and Lin(-)/CD45(-)/CD133(+) very small embryonic-like stem cells (VSELs), which express markers of the neural lineage, and also the plasma levels of factors that modulate their trafficking: the C3a, C5a, and C5b-9 activated complement cascade components, stromal-derived factor 1, and sphingosine-1-phosphate (S1P). We found that the mean numbers of Lin(-)/CD45(-)/CD34(+) VSELs and the plasma levels of S1P prior to treatment differ between the CG and PG and that these cells express markers of neural lineage. The number of Lin(-)/CD45(-)/CD133(+) VSELs in peripheral blood differed between the SG and NG prior to treatment. Using logistic regression analysis, we found that C3a and S1P are the best predictors of risk and are potential markers for the first psychotic episode. Furthermore, in the SG, the number of circulating Lin(-)/CD45(-)/CD34(+) VSELs and the S1P plasma level are the best predictors of risk and are proposed as novel markers for the first "schizophrenic" episode of psychosis.

Umbilical cord blood-derived very small embryonic like stem cells (VSELs) as a source of pluripotent stem cells for regenerative medicine.

Umbilical cord blood-derived very small embryonic-like stem cells (UCB-VSELs) are the most primitive stem cells circulating in fetal peripheral blood. These very rare cells slightly smaller than red blood cells i) become mobilized during delivery, ii) are enriched in fraction of CD133+ Lin-CD45- cells iii) express markers of pluripotent stem cells (e.g., Oct4, Nanog, and SSEA-4) and iv) display a distinct morphology characterized by a high nuclear/ cytoplasmic ratio and undifferentiated chromatin. We envision that VSELs are released into neonatal peripheral blood as a migrating population of stem cells involved in regeneration of tissues that become damaged in the process of delivery. They may also be responsible for the occurrence of fetal-maternal chimerism. Our most recent data suggest that UCB-VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells (LT-HSCs). We propose that UCB-VSELs may eventually be employed as a source of pluripotent stem cells in regenerative medicine.

Genomic architecture of alpha-amylase activity in mature rye grain relative to that of preharvest sprouting.

Bi-directional selective genotyping (BSG) carried out on two opposite groups of F(9)(541 × Ot1-3) recombinant inbred lines (RILs) with extremely low and extremely high alpha-amylase activities in mature (dry) grain of rye, followed by molecular mapping, revealed a complex system of selection-responsive loci. Three classes of loci controlling alpha-amylase activity were discerned, including four major AAD loci on chromosomes 3R (three loci) and 6RL (one locus) responding to both directions of the disruptive selection, 20 AAR loci on chromosomes 2RL (three loci), 3R (three loci), 4RS (two loci), 5RL (three loci), 6R (two loci) and 7R (seven loci) responding to selection for low alpha-amylase activity and 17 AAE loci on chromosomes 1RL (seven loci), 2RS (two loci), 3R (two loci), 5R (two loci) and 6RL (four loci) affected by selection for high alpha-amylase activity. The majority of the discerned AA loci also showed responsiveness to selection for preharvest sprouting (PHS). Two AAD loci on chromosome arm 3RL coincided with PHSD loci. The AAD locus on chromosome arm 3RS was independent from PHS, whereas that on chromosome 6RL belonged to the PHSR class. AAR-PHSR loci were found on chromosomes 4RS (one locus) and 5R (two loci) and AAE-PHSE loci were identified on chromosomes 1RL (one locus) and 5RL (one locus). Some PHSD loci represented the AAE (chromosomes 1RL, 3RS and 3RL) or AAR classes (chromosome 5RL). AAR and AAE loci not related to PHS were found on chromosomes 1RL, 2R, 3RS, 4R, 6RL and 7RL. On the other hand, several PHS loci (1RL, 3RS, 5RL, 6RS and 7RS) had no effect on alpha-amylase activity. Allele originating from the parental line 541 mapped in six AA loci on chromosomes 2R (two loci), 5R (three loci) and 7R (one locus) exerted opposite effects on PHS and alpha-amylase activity. Differences between the AA and PHS systems of loci may explain the weak correlation between these two traits observed among recombinant inbred lines. Strategies for the breeding of sprouting-resistant varieties with low alpha-amylase and high PHS resistance are discussed.