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Loss of mature β cell function and identity, or β cell dedifferentiation, is seen in both type 1 and type 2 diabetes. Two competing models explain β cell dedifferentiation in diabetes. In the first model, β cells dedifferentiate in the reverse order of their developmental ontogeny. This model predicts that dedifferentiated β cells resemble β cell progenitors. In the second model, β cell dedifferentiation depends on the type of diabetogenic stress. This model, which we call the “Anna Karenina” model, predicts that in each type of diabetes, β cells dedifferentiate in their own way, depending on how their mature identity is disrupted by any particular diabetogenic stress. We directly tested the two models using a β cell-specific lineage-tracing system coupled with RNA-sequencing in mice. We constructed a multidimensional map of β cell transcriptional trajectories during the normal course of β cell postnatal development and during their dedifferentiation in models of both type 1 diabetes (NOD) and type 2 diabetes (BTBR-Lepob/ob). Using this unbiased approach, we show here that despite some similarities between immature and dedifferentiated β cells, β cells dedifferentiation in the two mouse models is not a reversal of developmental ontogeny and is different between different types of diabetes.

Fatty acid-binding protein (FABP) 5 is highly expressed in various types of tumors and is strongly correlated with tumor growth, development, and metastasis. However, it is unclear how the expression of FABP5 in the host affects tumor progression. In this study, using a lung tumor metastasis model in mice, we found that FABP5-deficient mice were more susceptible to tumor metastasis, which is accompanied by infiltration of a lower frequency of activated natural killer (NK) cells in the lung. Additionally, FABP5 deficiency leads to impaired maturation of NK cells in the lungs, but not in the bone marrow and spleen. Taken together, our results provide the first evidence that FABP5 in the host regulates lung tumor metastasis through controlling NK cell maturation.

Macroenvironmental factors, including a patient’s physical and social environment, play a role in cancer risk and progression. Our previous preclinical studies have shown that the enriched environment (EE) confers anti-obesity and anti-cancer phenotypes that are associated with enhanced adaptive immunity and are mediated by brain-derived neurotrophic factor (BDNF). Natural killer (NK) cells have anti-cancer and anti-viral properties, and their absence or depletion is associated with inferior clinical outcomes. In this study, we investigated the effects of EE on NK cell maturation following their depletion. Mice living in EE displayed a higher proportion of NK cells in the spleen, bone marrow, and blood, compared to those living in the standard environment (SE). EE enhanced NK cell maturation in the spleen and was associated with upregulation of BDNF expression in the hypothalamus. Hypothalamic BDNF overexpression reproduced the EE effects on NK cell maturation in secondary lymphoid tissues. Conversely, hypothalamic BDNF knockdown blocked the EE modulation on NK cell maturation. Our results demonstrate that a bio-behavior intervention enhanced NK cell maturation and was mediated at least in part by hypothalamic BDNF.

Background Significant developments in stem cell therapy for Parkinson’s disease (PD) have already been achieved; however, methods for reliable assessment of dopamine neuron maturation in vivo are lacking. Establishing the efficacy of new cellular therapies using non-invasive methodologies will be critical for future regulatory approval and application. The current study examines the utility of neuroimaging to characterise the in vivo maturation, innervation and functional dopamine release of transplanted human embryonic stem cell-derived midbrain dopaminergic neurons (hESC-mDAs) in a preclinical model of PD. Methods Female NIH RNu rats received a unilateral stereotaxic injection of 6-OHDA into the left medial forebrain bundle to create the PD lesion. hESC-mDA cell and sham transplantations were carried out 1 month post-lesion, with treated animals receiving approximately 4 × 105 cells per transplantation. Behavioural analysis, [18F]FBCTT and [18F]fallypride microPET/CT, was conducted at 1, 3 and 6 months post-transplantation and compared with histological characterisation at 6 months. Results PET imaging revealed transplant survival and maturation into functional dopaminergic neurons. [18F]FBCTT-PET/CT dopamine transporter (DAT) imaging demonstrated pre-synaptic restoration and [18F]fallypride-PET/CT indicated functional dopamine release, whilst amphetamine-induced rotation showed significant behavioural recovery. Moreover, histology revealed that the grafted cells matured differently in vivo producing high- and low-tyrosine hydroxylase (TH) expressing cohorts, and only [18F]FBCTT uptake was well correlated with differentiation. Conclusions This study provides further evidence for the value of in vivo functional imaging for the assessment of cell therapies and highlights the utility of DAT imaging for the determination of early post-transplant cell maturation and differentiation of hESC-mDAs.

Therapeutic hypothermia is now well established to partially reduce disability in term and near-term infants with moderate-severe hypoxic-ischemic encephalopathy. Preclinical and clinical studies have confirmed that current protocols for therapeutic hypothermia are near optimal. The challenge is now to identify complementary therapies that can further improve outcomes, in combination with therapeutic hypothermia. Overall, anti-excitatory and anti-apoptotic agents have shown variable or even no benefit in combination with hypothermia, suggesting overlapping mechanisms of neuroprotection. Inflammation appears to play a critical role in the pathogenesis of injury in the neonatal brain, and thus, there is potential for drugs with immunomodulatory properties that target inflammation to be used as a therapy in neonates. In this review, we examine the evidence for neuroprotection with immunomodulation after hypoxia-ischemia. For example, stem cell therapy can reduce inflammation, increase cell survival, and promote cell maturation and repair. There are also encouraging preclinical data from small animals suggesting that stem cell therapy can augment hypothermic neuroprotection. However, there is conflicting evidence, and rigorous testing in translational animal models is now needed.

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo., Identifying how genetic alterations cooperate in cancer is challenging. Here the authors analyze leukemia mouse models with both oncogenic NRAS and EZH2 mutations using single-cell RNA-sequencing, evaluate oncogenic cooperation, and identify GEM as a regulator of leukemia-initiating cells.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and possess considerable immunomodulatory potential. MSCs have been shown to promote allograft survival, yet the mechanisms behind this phenomenon have not been fully defined. Here, we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation, we report that MSCs promote graft survival in an HGF-dependent manner. Moreover, our data indicate that topically administered recombinant HGF (a) suppresses antigen-presenting cell maturation in draining lymphoid tissue, (b) limits T-helper type-1 cell generation, (c) decreases inflammatory cell infiltration into grafted tissue, and (d) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019;8:1030–1040

Increasing evidence demonstrates that IL-17A promotes tumorigenesis, metastasis, and viral infection. Natural killer (NK) cells are critical for defending against tumors and infections. However, the roles and mechanisms of IL-17A in regulating NK cell activity remain elusive. Herein, our study demonstrated that IL-17A constrained NK cell antitumor and antiviral activity by restraining NK cell maturation. It was observed that the development and metastasis of tumors were suppressed in IL-17A–deficient mice in the NK cell-dependent manner. In addition, the antiviral activity of NK cells was also improved in IL-17A–deficient mice. Mechanistically, ablation of IL-17A signaling promoted generation of terminally mature CD27(−)CD11b(+) NK cells, whereas constitutive IL-17A signaling reduced terminally mature NK cells. Parabiosis or mixed bone marrow chimeras from Il17a(−/−)and wild-type (WT) mice could inhibit excessive generation of terminally mature NK cells induced by IL-17A deficiency. Furthermore, IL-17A desensitized NK cell responses to IL-15 and suppressed IL-15–induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) via up-regulation of SOCS3, leading to down-regulation of Blimp-1. Therefore, IL-17A acts as the checkpoint during NK cell terminal maturation, which highlights potential interventions to defend against tumors and viral infections.

BackgroundMicrobial colonization and immune cell maturation coincide at mucosal sites and are decisive for postnatal lung development. How external factors influence neonatal pulmonary immune development is poorly understood.ObjectiveTo elucidate the impact of key determinants in early life, nutrition, and maternal bonding, on postnatal lung maturation in a human-relevant animal model. To investigate the underlying immunological changes of impaired lung maturation and study the mechanisms of conversion.MethodsNewborn piglets were kept with or without isolation from their mothers and fed bovine milk-based infant formula or received milk of sow. Lung growth, histomorphology, respiratory immune responses, and lung microbiota were analyzed. Mother- and sow-milk-deprived piglets received maternal material or were reintroduced to the maternal environment at varying intervals to study options for reversal.ResultsFormula feeding combined with isolation of newborn piglets resulted in disturbed postnatal lung maturation. Reduced lung growth correlated with dampened IL-33 expression, impaired lung myeloid cell activation, and decreased Th1 differentiation, along with diminished richness and diversity of the lung microbiota. Transfer of bacteria-enriched maternal material reversed the negative effects on pulmonary immune maturation. Early (within 3 days) but not late (within 7 days) reintroduction to the mother allowed restoration of normal lung development.ConclusionOur findings reveal that lung growth, respiratory immunity, and microbial lung colonization in newborns depend on postnatal diet and maternal contact, and targeting these key regulators could promote lung development during this critical life stage.SummaryDisturbances in natural diet and reduced maternal contact during the neonatal period impair postnatal lung maturation. In pediatrics, timely breast milk feeding and intensive maternal bonding represent valuable intervention measures to promote early postnatal lung development.

The coronavirus disease 2019 (COVID-19) pandemic had significant impact on research directions and productivity in the past year. Despite these challenges, since 2020, >2,500 peer-reviewed articles in pancreatic islet biology have been published. These include updates on the roles of isocitrate dehydrogenase, pyruvate kinase, and incretin hormones in insulin secretion, as well as the discovery of Inceptor and signaling by circulating RNAs. 2020 also brought advancements in in vivo and in vitro models, including a new transgenic mouse for assessing β-cell proliferation, a pancreas-on-a-chip to study glucose-stimulated insulin secretion, and successful genetic editing of primary human islet cells. Islet biologists evaluated the functionality of stem cell-derived islet-like cells coated with semi-permeable biomaterials to prevent autoimmune attack, revealing the importance of cell maturation following transplantation. Prompted by observations that COVID-19 symptoms can worsen for people with obesity or diabetes, researchers examined how islets are directly affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, we highlight novel functional insights, technologies and therapeutic approaches emerging between March 2020 and July 2021, written for both scientific and lay audiences. We also include a response to these advancements from patient stakeholders, to help lend broader perspective to developments and challenges in islet research., Graphical abstract

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies. ispartof: FRONTIERS IN IMMUNOLOGY vol:12 ispartof: location:Switzerland status: published

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape “fibro-chondrocytic” cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.

In adult dentate gyrus neurogenesis, the link between maturation of newborn neurons and their function, such as behavioral pattern separation, has remained puzzling. By analyzing a theoretical model, we show that the switch from excitation to inhibition of the GABAergic input onto maturing newborn cells is crucial for their proper functional integration. When the GABAergic input is excitatory, cooperativity drives the growth of synapses such that newborn cells become sensitive to stimuli similar to those that activate mature cells. When GABAergic input switches to inhibitory, competition pushes the configuration of synapses onto newborn cells toward stimuli that are different from previously stored ones. This enables the maturing newborn cells to code for concepts that are novel, yet similar to familiar ones. Our theory of newborn cell maturation explains both how adult-born dentate granule cells integrate into the preexisting network and why they promote separation of similar but not distinct patterns.

Glucose homeostasis depends on regulated insulin secretion from pancreatic β cells, which acquire their mature phenotype postnatally. The functional maturation of β cells is regulated by a combination of cell-autonomous and exogenous factors; the identity of the latter is mostly unknown. Here, we identify BMP4 as a critical component through which the pancreatic microenvironment regulates β cell function. By combining transgenic mouse models and human iPSCs, we show that BMP4 promotes the expression of core β cell genes and is required for proper insulin production and secretion. We identified pericytes as the primary pancreatic source of BMP4, which start producing this ligand midway through the postnatal period, at the age β cells mature. Overall, our findings show that the islet niche directly promotes β cell functional maturation through the timely production of BMP4. Our study highlights the need to recapitulate the physiological postnatal islet niche for generating fully functional stem-cell-derived β cells for cell replacement therapy for diabetes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in ‘cell development’, ‘cell growth’, ‘cell differentiation’ and ‘cell maturation’ processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Transfer-RNAs (tRNAs) help ribosomes decode mRNAs and synthesize proteins; however, tRNA fragments produced under certain conditions, known as tRNA-derived small RNAs (tsRNAs), have been found to play important roles in pathophysiological processes. In the reproductive system, tsRNAs are abundant in gametes and embryos and at the maternal–fetal interface, as well as in microvesicles like epididymosomes, seminal plasma exosomes, and syncytiotrophoblast-derived extracellular vesicles. tsRNAs can affect gamete cell maturation, zygote activation, and early embryonic development. tsRNAs can transmit epigenetic information to later generations. In particular, exposure to environmental factors such as nutrition, isoproterenol, and poly(I:C) may allow tsRNAs to transfer information to the gametes or placenta to alter offspring phenotype. The underlying mechanisms of tsRNAs action include transposon silencing, translation regulation, and target mRNA degradation. Herein, we review the currently reported tsRNAs in the reproductive system, their validated functions, and potential roles. A better understanding of this field may help to provide useful recommendations or develop strategies to increase fertility and conception of healthy babies.

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) causes a rare and debilitating form of X-linked psychomotor disability known as Allan Herndon Dudley syndrome (AHDS). One of the most prominent pathophysiological symptoms of MCT8-deficiency is hypomyelination. Here, patient-derived induced pluripotent stem cells (iPSCs) were used to study the role of MCT8 and TH on the maturation of oligodendrocytes. Interestingly, neither MCT8 mutations nor reduced TH affected the in vitro differentiation of control or MCT8-deficient iPSCs into oligodendrocytes. To assess whether patient-derived iPSC-derived oligodendrocyte progenitor cells (iOPCs) could provide myelinating oligodendrocytes in vivo, cells were transplanted into the shiverer mouse corpus callosum where they survived, migrated, and matured into myelinating oligodendrocytes, though the myelination efficiency was reduced compared with control cells. When MCT8-deficient and healthy control iOPCs were transplanted into a novel hypothyroid immunodeficient triple knockout mouse (tKO, mct8-/- ; oatp1c1-/- ; rag2-/- ), they failed to provide behavioral recovery and did not mature into oligodendrocytes in the hypothyroid corpus callosum, demonstrating the critical role of TH transport across brain barriers in oligodendrocyte maturation. We conclude that MCT8 plays a cell autonomous role in oligodendrocyte maturation and that functional TH transport into the central nervous system will be required for developing an effective treatment for MCT8-deficient patients.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1β isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1β (Stat1β/β ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1β/β mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1β/β mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1β/β mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

Cellular differentiation is a tightly regulated process under the control of intricate signaling and transcription factors interaction network working in coordination. These interactions make the systems dynamic, robust and stable but also difficult to dissect. In the spinal cord, recent work has shown that a network of FGF, WNT and Retinoic Acid (RA) signaling factors regulate neural maturation by directing the activity of a transcription factor network that contains CDX at its core. Here we have used partial and ordinary (Hill) differential equation based models to understand the spatiotemporal dynamics of the FGF/WNT/RA and the CDX/transcription factor networks, alone and in combination. We show that in both networks, the strength of interaction among network partners impacts the dynamics, behavior and output of the system. In the signaling network, interaction strength determine the position and size of discrete regions of cell differentiation and small changes in the strength of the interactions among networking partners can result in a signal overriding, balancing or oscillating with another signal. We also show that the spatiotemporal information generated by the signaling network can be conveyed to the CDX/transcription network to produces a transition zone that separates regions of high cell potency from regions of cell differentiation, in agreement with mostin vivoobservations. Importantly, one emerging property of the networks is their robustness to extrinsic disturbances, which allows the system to retain or canalize NP cells in developmental trajectories. This analysis provides a model for the interaction conditions underlying spinal cord cell maturation during embryonic axial elongation.

The MYC oncogene drives T- and B- lymphoid malignancies, including Burkitt’s lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Here, we demonstrate a systemic reduction in natural killer (NK) cell numbers in SRα-tTA/Tet-O-MYCON mice bearing MYC-driven T-lymphomas. Residual mNK cells in spleens of MYCON T-lymphoma-bearing mice exhibit perturbations in the terminal NK effector differentiation pathway. Lymphoma-intrinsic MYC arrests NK maturation by transcriptionally repressing STAT1/2 and secretion of Type I Interferons (IFNs). Treating T-lymphoma-bearing mice with Type I IFN improves survival by rescuing NK cell maturation. Adoptive transfer of mature NK cells is sufficient to delay both T-lymphoma growth and recurrence post MYC inactivation. In MYC-driven BL patients, low expression of both STAT1 and STAT2 correlates significantly with the absence of activated NK cells and predicts unfavorable clinical outcomes. Our studies thus provide a rationale for developing NK cell-based therapies to effectively treat MYC-driven lymphomas in the future., Oncogene addiction is considered as a cancer cell-autonomous phenomenon, but can also influence the host immune system. Here the authors show that MYC-driven lymphomagenesis is associated with a block in the maturation and effector functions of natural killer cells as a mechanism of tumor escape from immunosurveillance.

Previous studies of NK cell inhibitory Ly49 receptors suggested their expression is stochastic. However, relatively few studies have examined this stochasticity in conjunction with activating Ly49 receptors. We hypothesized that the expression of activating Ly49 receptors is not stochastic and is influenced by inhibitory Ly49 receptors. We analyzed NK cell “clusters” defined by combinatorial expression of activating (Ly49H, Ly49D) and inhibitory (Ly49I, Ly49G2) receptors in C57BL/6 mice. Using the product rule to evaluate the interdependencies of the Ly49 receptors, we found evidence for a tightly regulated expression at the immature NK cell stage, with the highest interdependencies between clusters that express at least one activating receptor. Further analysis demonstrated that certain NK clusters predominated at the immature (CD27+CD11b−), transitional (CD27+CD11b+) and mature (CD27−CD11b−) NK cell stages. Using parallel in vitro culture and in vivo transplantation of sorted NK clusters, we discovered non-random upregulation of Ly49 receptors, suggesting that prescribed pathways of NK cluster differentiation exist. Our data infer that upregulation of Ly49I is an important step in NK cell maturation. Ki-67 expression and cell counts confirmed that immature NK cells proliferate more than mature NK cells. We found that MHC-I is particularly important for regulation of Ly49D and Ly49G2, even though no known MHC-I ligand for these receptors is present in B6 mice. Our data indicate that the regulatory systems controlling the expression of both activating and inhibitory Ly49 receptors are non-stochastic and support the idea that NK cell clusters develop in a non-random process correlated to their maturation stage.

Today, many attempts have been collected in the field of tissue engineering for reconstitution of injured bone marrow capacity by transplantation of functional cell source. By having a three-dimensional condition, microcapsules are appropriate candidates for cells transplantation to target sites. Here, we examined the effect of alginate-gelatin microcapsules on functional maturation of human myelomonocytic cell line U937 after 7 days in vitro. U937 cells were encapsulated by the mixture of alginate-gelatin and cultured for 7 days. Trypan blue staining was used to show cell survival rate. Morphological changes were determined by haematoxylin and eosin staining. The expression of monocyte (CD14) and leukocyte (CD33) factors were measured by flow cytometry. The functional maturation of encapsulated cells was shown by immunocytochemistry targeting myeloperoxidase (MPO) activity and level of CD68. Transcription level of adhesion molecules CD68L, CD18, CD11b, and CD49d/VLA was detected by real-time polymerase chain reaction. In vivo constitutive capacity of encapsulated U937 was investigated in rabbits via administration to bone marrow. We showed enhanced U937 viability and monocyte and band cell-like appearance 7 days after encapsulation. These changes coincided with increasing CD33 and CD14 levels and a decrease of CD15, confirming cell maturation (p < 0.05). High level of MPO and CD68-positive cells showed the functional maturation of U937 cells into neutrophils and macrophages. Compared with that of nonencapsulated cells, the level of adhesion factor was up-regulated. We found labelled cells in the peripheral blood after cell transplantation to bone marrow. These data suggest that alginate-gelatin encapsulation of U937 cells promotes functional leukopoiesis and monocytopoiesis.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions., The metabolic regulator protein family, mTOR, regulate natural killer (NK) cell development and function, but the underlying mechanism is unclear. Here, the authors show that Raptor/mTORC1 and Rictor/mTORC2 form a feedback crosstalk network to variegate cytokine and cellular signaling and modulate NK maturation and effector functions.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization delayed in Col2Cre(+);Dnmt3b(f/f) (Dnmt3b(Col2)) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3b(Col2) mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3b(Col2) growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3b(Col2) mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3b(Col2) chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

Development of cell replacement therapies in diabetes requires understanding of the molecular underpinnings of β-cell maturation. The circadian clock regulates diverse cellular functions important for regulation of β-cell function and turnover. However, postnatal ontogenesis of the islet circadian clock and its potential role in β-cell maturation remain unknown. To address this, we studied wild-type Sprague-Dawley as well as Period1 luciferase transgenic (Per1:LUC) rats to determine circadian clock function, clock protein expression, and diurnal insulin secretion during islet development and maturation process. We additionally studied β-cell–specific Bmal1-deficient mice to elucidate a potential role of this key circadian transcription factor in β-cell functional and transcriptional maturation. We report that emergence of the islet circadian clock 1) occurs during the early postnatal period, 2) depends on the establishment of global behavioral circadian rhythms, and 3) leads to the induction of diurnal insulin secretion and gene expression. Islet cell maturation was also characterized by induction in the expression of circadian transcription factor BMAL1, deletion of which altered postnatal development of glucose-stimulated insulin secretion and the associated transcriptional network. Postnatal development of the islet circadian clock contributes to early-life β-cell maturation and should be considered for optimal design of future β-cell replacement strategies in diabetes.

The preterm newborn is at significant risk of neural injury and impaired neurodevelopment. Infants with mild or no evidence of injury may also be at risk of altered brain development, with evidence impaired cell maturation. The underlying causes are multifactorial and include exposure of both the fetus and newborn to hypoxia-ischemia, inflammation (chorioamnionitis) and infection, adverse maternal lifestyle choices (smoking, drug and alcohol use, diet) and obesity, as well as the significant demand that adaptation to post-natal life places on immature organs. Further, many fetuses and infants may have combinations of these events, and repeated (multi-hit) events that may induce tolerance to injury or sensitize to greater injury. Currently there are no treatments to prevent preterm injury or impaired neurodevelopment. However, inflammation is a common pathway for many of these insults, and clinical and experimental evidence demonstrates that acute and chronic inflammation is associated with impaired brain development. This review examines our current knowledge about the relationship between inflammation and preterm brain development, and the potential for stem cell therapy to provide neuroprotection and neurorepair through reducing inflammation and release of trophic factors, which promote cell maturation and repair.

SUMMARY Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications., Graphical Abstract, In Brief May-Simera et al. show that primary cilia regulate the maturation and polarization of human iPSC-RPE, mouse RPE, and human iPSC-lung epithelium through canonical WNT suppression and PKCδ activation. RPE cells derived from ciliopathy patients exhibit defective structure and function. These results provide insights into ciliopathy-induced retinal degeneration.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

The leukocyte immunoglobulin-like receptor (LILR) family comprises a set of paired immunomodulatory receptors expressed among human myeloid and lymphocyte cell populations. While six members of LILR subfamily A (LILRA) associate with membrane adaptors to signal via immunoreceptor tyrosine-based activating motifs (ITAM), LILR subfamily B (LILRB) members signal via multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). Ligand specificity of some LILR family members has been studied in detail, but new perspective into the immunoregulatory aspects of this receptor family in human myeloid cells has been limited. LILRB receptors and the murine ortholog, paired immunoglobulin-like receptor B (PIRB), have been shown to negatively regulate maturation pathways in myeloid cells including mast cells, neutrophils, dendritic cells, as well as B cells. Our laboratory further demonstrated in mouse models that PIRB regulated functional development of myeloid-derived suppressor cell and the formation of a tumor-permissive microenvironment. Based on observations from the literature and our own studies, our laboratory is focusing on how LILRs modulate immune homeostasis of human myeloid cells and how these pathways may be targeted in disease states. Integrity of this pathway in tumor microenvironments, for example, permits a myeloid phenotype that suppresses antitumor adaptive immunity. This review presents the evidence supporting a role of LILRs as myeloid cell regulators and ongoing efforts to understand the functional immunology surrounding this family.

SummaryMammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.

PURPOSE OF REVIEW This report examines recent publications identifying phenotypic and functional heterogeneity among pancreatic β cells and investigating their potential roles in normal and abnormal islet function. The development of new methods and tools for the study of individual islet cells has produced a surge of interest in this topic. RECENT FINDINGS Studies of β cell maturation and pregnancy-induced proliferation have identified changes in serotonin and transcription factors SIX2/3 expression as markers of temporal heterogeneity. Structural and functional heterogeneity in the form of functionally distinct 'hub' and 'follower' β cells was found in mouse islets. Heterogeneous expression of Fltp (in mouse β cells) and ST8SIA1 and CD9 (in human β cells) were associated with distinct functional potential. Several impressive reports describing the transcriptomes of individual β cells were also published in recent months. Some of these reveal previously unknown β cell subpopulations. SUMMARY A wealth of information on functional and phenotypic heterogeneity has been collected recently, including the transcriptomes of individual β cells and the identities of functionally distinct β cell subpopulations. Several studies suggest the existence of two broad categories: a more proliferative but less functional and a less proliferative but more functional β cell type. The identification of functionally distinct subpopulations and their association with type 2 diabetes underlines the potential clinical importance of these investigations.

Multiple intra-cellular signalling pathways rely on calcium and 3′–5′ cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.

Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer&rsquo, s disease (AD) patients and animal AD models, however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administered an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and applied an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidenced a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline showed no significant effect upon the progenitor cell number and constitution. We demonstrated that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identified a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.

Summary Differentiation of human embryonic stem cells into pancreatic β cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary β cell counterparts. The barrier(s) to in vitro β cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated β cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward β cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of β cell maturation in vitro., Graphical Abstract, Highlights • Let-7 is low in hESC-derived relative to in vivo matured human β cells • LIN28 is elevated in hESC-derived relative to in vivo matured human islet β cells • Depletion of LIN28, but not gain of let-7, promotes hESC-derived β cell maturation • Depletion of LIN28 promotes silencing of juvenile-specific transcriptional program, In this article, Blelloch, Hebrok, and colleagues show that in vitro hESC-derived islet β cells express elevated LIN28 and reduced let-7 levels relative to their in vivo matured counterparts. Reduction of LIN28, but not increase in let-7, promotes suppression of a juvenile-specific β cell transcriptional program, implicating LIN28 as a barrier to β cell maturation in vitro.

β Cell apoptosis and dedifferentiation are 2 hotly debated mechanisms underlying β cell loss in type 2 diabetes; however, the molecular drivers underlying such events remain largely unclear. Here, we performed a side-by-side comparison of mice carrying β cell–specific deletion of ER-associated degradation (ERAD) and autophagy. We reported that, while autophagy was necessary for β cell survival, the highly conserved Sel1L-Hrd1 ERAD protein complex was required for the maintenance of β cell maturation and identity. Using single-cell RNA-Seq, we demonstrated that Sel1L deficiency was not associated with β cell loss, but rather loss of β cell identity. Sel1L-Hrd1 ERAD controlled β cell identity via TGF-β signaling, in part by mediating the degradation of TGF-β receptor 1. Inhibition of TGF-β signaling in Sel1L-deficient β cells augmented the expression of β cell maturation markers and increased the total insulin content. Our data revealed distinct pathogenic effects of 2 major proteolytic pathways in β cells, providing a framework for therapies targeting distinct mechanisms of protein quality control.

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.

Germinal centers (GCs) are transient anatomical microenvironments where antibody affinity maturation and memory B cells generation takes place. In the past, models of Germinal Center (GC) dynamics have focused on understanding antibody affinity maturation rather than on the main mechanism(s) driving their rise-and-fall dynamics. Here, based on a population dynamics model core, we compare three mechanisms potentially responsible for this GC biphasic behavior dependent on follicular dendritic cell (FDC) maturation, follicular T helper (Tfh) cell maturation, and antigen depletion. Analyzing the kinetics of B and T cells, as well as its parameter sensitivities, we found that only the FDC-maturation-based model could describe realistic GC dynamics, whereas the simple Tfh-maturation and antigen-depletion mechanisms, as implemented here, could not. We also found that in all models the processes directly related to Tfh cell kinetics have the highest impact on GC dynamics. This suggests the existence of some still unknown mechanism(s) tuning GC dynamics by affecting Tfh cell response to proliferation-inducing stimuli. Fundação para Ciência e Tecnologia | Ref. POCTI/36413/1999 European Commission | Ref. REGPOT-2012-2013.1-316265 (BIOCAPS) European Commission | Ref. PIRSES-GA-2012-317893 (INDOEUROPEAN-MATHDS) Xunta de Galicia | Ref. ED431C 2016041

International audience; The respiratory epithelium arises from alveolar epithelial progenitors which differentiate into alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. AT2 cells are stem cells in the lung critical for the repair process after injury. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report that the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using miR-142 KO mice, we demonstrate an increase in the AT2/AT1 cell number ratio. Overexpression of miR-142 in alveolar progenitor cells in vivo led to the opposite effect. Examination of the KO lungs at E18.5 revealed enhanced expression of miR-142 targets Apc, Ep300 and Kras associated with increased β-catenin and p-Erk signaling. Silencing of miR-142 expression in lung explants grown in vitro triggers enhanced Sftpc expression as well as increased AT2/AT1 cell number ratio. Pharmacological inhibition of Ep300-β-catenin but not Erk in vitro prevented the increase in Sftpc expression triggered by loss of miR-142. These results suggest that the glucocorticoid-miR-142-Ep300-β-catenin signaling axis controls pneumocyte maturation.

NK cell maturation is a continuous process, which initiates in the bone marrow and proceeds in peripheral tissues, where NK cells follow distinct differentiation routes. Drastic phenotypic changes are observed during progression from precursors to mature NK cells, including changes of expression and functionalities of several chemoattractant receptors. Upon differentiation, mature NK cells migrate outside the bone marrow; as well, peculiar subsets of NK cells can also home back to or localize in this anatomic compartment to play specific functions. In humans, NK cells with a tissue resident phenotype have been identified in bone marrow, sharing similarities with tissue resident memory CD8+ T cells; while in mouse, long-lived NK cells undergo homeostatic proliferation in this site during viral infections. The mechanisms underlying NK cell subset localization in the bone marrow have only recently started to be investigated, especially in pathological settings such as tumors or infections. In this review, we discuss the phenotype and function of NK cells as well as their requirements for bone marrow maintenance and/or homing.

Na-K-ATPase on the basolateral membrane provides the favorable transcellular Na gradient for the proper functioning of Na-dependent nutrient co-transporters on the brush border membrane (BBM) of enterocytes. As cells mature from crypts to villus, Na-K-ATPase activity doubles, to accommodate for the increased BBM Na-dependent nutrient absorption. However, the mechanism of increased Na-K-ATPase activity during the maturation of enterocytes is not known. Therefore, this study aimed to determine the mechanisms involved in the functional transition of Na-K-ATPase during the maturation of crypts to villus cells. Na-K-ATPase activity gradually increased as IEC-18 cells matured in vitro from day 0 (crypts) through day 4 (villus) of post-confluence. mRNA abundance and Western blot studies showed no change in the levels of Na-K-ATPase subunits &alpha, 1 and &beta, 1 from 0 to 4 days post-confluent cells. However, Na-K-ATPase &alpha, 1 phosphorylation levels on serine and tyrosine, but not threonine, residues gradually increased. These data indicate that as enterocytes mature from crypt-like to villus-like in culture, the functional activity of Na-K-ATPase increases secondary to altered affinity of the &alpha, 1 subunit to extracellular K+, in order to accommodate the functional preference of the intestinal cell type. This altered affinity is likely due to increased phosphorylation of the &alpha, 1 subunit, specifically at serine and tyrosine residues.

Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24–48 hours, Ag-specific B cells were detectable at T–B borders in the spleen; there, they proliferated in a T cell–dependent manner and induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH cells after CD180 targeting: TFH cells were induced in BAFFR−/− mice (with only transitional 1 B cells) and not in μMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, providing insights toward vaccination of mature B cell–deficient individuals and implications in treating autoimmune disorders.

Natural killer (NK) cells are the prototypical members of the recently identified family of innate lymphoid cells (ILCs). Thanks to their cytotoxic and secretory functions, NK cells play a key role in the immune response to cells experiencing various forms of stress, including viral infection and malignant transformation. Autophagy is a highly conserved network of degradative pathways that participate in the maintenance of cellular and organismal homeostasis as they promote adaptation to adverse microenvironmental conditions. The relevance of autophagy in the development and functionality of cellular components of the adaptive immune system is well established. Conversely, whether autophagy also plays an important role in the biology of ILC populations such as NK cells has long remained elusive. Recent experimental evidence shows that ablating Atg5 (autophagy-related 5, an essential component of the autophagic machinery) in NK cells and other specific ILC populations results in progressive mitochondrial damage, reactive oxygen species (ROS) overgeneration, and regulated cell death, hence interrupting ILC development. Moreover, disrupting the interaction of ATG7 with phosphorylated FOXO1 (forkhead box O1) in the cytosol of immature NK cells prevents autophagic responses that are essential for NK cell maturation. These findings suggest that activating autophagy may support the maturation of NK cells and other ILCs that manifest antiviral and anticancer activity.

Using PrPC-knockout cell lines, it has been shown that the inhibition of apoptosis through STI1 is mediated by PrPC-dependent SOD activation. Antioxidant PrPC may contribute to suppression of inflammasome activation. PrPC is functionally involved in copper metabolism, signal transduction, neuroprotection, and cell maturation. Recently several reports have shown that PrPC participates in trans-membrane signaling processes associated with hematopoietic stem cell replication and neuronal differentiation. In another role, PrPC also tends to function as a neurotoxic protein. Aβ oligomer, which is associated with neurodegeneration in Alzheimer’s disease (AD), has also been reported to act as a ligand of PrPC. However, the physiological role of PrPC as an Aβ42-binding protein is not clear. Actually, PrPC is critical in Aβ42-mediated autophagy in neurons. PrPC shows a beneficial role in lipid rafts to promote autophagy. Further search for PrPC-interaction molecules using Prnp−/− mice and various types of Prnp−/− cell lines under various conditions may elucidate other important PrPC important functions.

According to Ming's classification, gastric cancer (GC) can be divided into two types: expanding and infiltrative. The two types are readily recognizable by histology: expanding carcinomas grow en masse and by expansion, resulting in the formation of discrete tumour nodules, whereas in infiltrative carcinoma, tumour cells invade individually. Both types show varying degrees of cell maturation. The two types of carcinomas have vastly different pathological and clinical features. However, little is known concerning the mechanisms underlying these differences since no GC cell line models are available. For comprehensive and insightful analyses of mechanisms and treatment methods, new cell lines derived from expanding- and infiltrative-type gastric tumours are urgently needed. In the present study, we established an expanding-type GC cell line from a 72-year-old male patient. Different in vitro and in vivo methods were used to characterize the phenotypes of this cell line. This GC cell line was named XGC-2 and had an ~60 h doubling time. The cell line displayed strong colony formation and tumourigenicity in nude mice and had complicated chromosomal abnormalities. XGC-2 cells showed some markers of epithelial-to-mesenchymal transition (EMT), with decreased E-cadherin expression levels and increased vimentin expression levels. The XGC-2 cell line may be useful for future studies of GC development, progression, metastasis and therapy.

Chronic stress significantly affects neuron morphometry and function in the prefrontal cortex, a brain region controlling cognition and emotion. However, whether and how chronic stress regulates the maturation of NG2-expressing oligodendrocyte precursor cell (NG2(+) cell) and the importance of these changes remained unknown. Here, we report that exposing adult mice to chronic stress results in NG2(+) cell atrophy and myelination arrested in the medial prefrontal cortex (mPFC), and impaired mPFC-dependent functions. These alterations, are phenocopied by overexpression of death receptor 6 (DR6) in NG2(+) cell. Conversely, selectively silencing of DR6 in the NG2(+) cell can partly rescue NG2(+) cell atrophy and cognitive deficiency caused by chronic stress. We further demonstrate that myelination appears necessary for mPFC-dependent cognitive processes, as lysolecithin (LPC)-induced demyelination specifically in the mPFC is sufficient to cause these behavioral and cognitive impairments. Our results indicate that chronic stress impairs cognitive functions, at least in part, through modulation of NG2(+) cell maturation and myelination, and suggest that myelination is require for normal cognitive functions.

Stromal cells (SCs) are strategically positioned in both lymphoid and nonlymphoid organs to provide a scaffold and orchestrate immunity by modulating immune cell maturation, migration and activation. Recent characterizations of SCs have expanded our understanding of their heterogeneity and suggested a functional specialization of distinct SC subsets, further modulated by the microenvironment. Lymph node SCs (LNSCs) have been shown to be particularly important in maintaining immune homeostasis and T cell tolerance. Under inflammation situations, such as viral infections or tumor development, SCs undergo profound changes in their numbers and phenotype and play important roles in contributing to either the activation or the control of T cell immunity. In this review, we highlight the role of SCs located in LNs in shaping peripheral T cell responses in different immune contexts, such as autoimmunity, viral and cancer immunity.

Pancreatic β-cells are responsible for production and secretion of insulin in response to increasing blood glucose levels. Defects in β-cell function lead to hyperglycemia and diabetes mellitus. Here, we show that CNOT3, a CCR4–NOT deadenylase complex subunit, is dysregulated in islets in diabetic db/db mice, and that it is essential for murine β cell maturation and identity. Mice with β cell-specific Cnot3 deletion (Cnot3βKO) exhibit impaired glucose tolerance, decreased β cell mass, and they gradually develop diabetes. Cnot3βKO islets display decreased expression of key regulators of β cell maturation and function. Moreover, they show an increase of progenitor cell markers, β cell-disallowed genes, and genes relevant to altered β cell function. Cnot3βKO islets exhibit altered deadenylation and increased mRNA stability, partly accounting for the increased expression of those genes. Together, these data reveal that CNOT3-mediated mRNA deadenylation and decay constitute previously unsuspected post-transcriptional mechanisms essential for β cell identity., Dina Mostafa et al. report that β cell function and identity depends on the deadenylase complex subunit CNOT3. The authors found that Cnot3 was dysregulated in diabetic mice and, when knocked out in wild type, mice have impaired glucose tolerance and gradually develop diabetes.

Paracrine erythropoietin (EPO) signaling in the lung recruits endothelial progenitor cells, promotes cell maturation and angiogenesis, and is upregulated during canine postpneumonectomy (PNX) compensatory lung growth. To determine whether inhalational delivery of exogenous EPO augments endogenous post-PNX lung growth, adult canines underwent right PNX and received, via a permanent tracheal stoma, weekly nebulization of recombinant human EPO-containing nanoparticles or empty nanoparticles (control) for 16 wk. Lung function was assessed under anesthesia pre- and post-PNX. The remaining lobes were fixed for detailed morphometric analysis. Compared with control treatment, EPO delivery significantly increased serum EPO concentration without altering systemic hematocrit or hemoglobin concentration and abrogated post-PNX lipid oxidative stress damage. EPO delivery modestly increased post-PNX volume densities of the alveolar septum per unit of lung volume and type II epithelium and endothelium per unit of septal tissue volume in selected lobes. EPO delivery also augmented the post-PNX increase in alveolar double-capillary profiles, a marker of intussusceptive capillary formation, in all remaining lobes. EPO treatment did not significantly alter absolute resting lung volumes, lung and membrane diffusing capacities, alveolar-capillary blood volume, pulmonary blood flow, lung compliance, or extravascular alveolar tissue volumes or surface areas. Results established the feasibility of chronic inhalational delivery of growth-modifying biologics in a large animal model. Exogenous EPO selectively enhanced cytoprotection and alveolar angiogenesis in remaining lobes but not whole-lung extravascular tissue growth or resting function; the nonuniform response contributes to structure-function discrepancy, a major challenge for interventions aimed at amplifying the innate potential for compensatory lung growth.