The transcription factors Junb and Fosl2 cooperate to regulate Cdh3 expression in 15P-1 Sertoli cells.

In Sertoli cells of the testis, cadherins (Cdh) are important cell-to-cell interaction proteins and contribute to the formation of the blood-testis barrier being essential for germ cells' protection. P-cadherin or Cdh3 is only expressed in Sertoli cells from embryonic to prepubertal development. Interestingly, the expression profile of Cdh3 correlates with that of activating protein-1 (AP-1) transcription factors during Sertoli cells development. To assess their potential implications in the regulation of Cdh3, different AP-1 transcription factors were overexpressed in 15P-1 Sertoli cells. We found that the overexpressions of Junb and Fosl2 activated Cdh3 promoter. ChIP-qPCR assay and luciferase reporter assay with 5' promoter deletions and site-directed mutagenesis confirmed the recruitment of Junb and Fosl2 to an AP-1 regulatory element at -47 bp in the proximal region of Cdh3 promoter in 15P-1 cells. These findings were further supported by histone modification markers and chromatin accessibility surrounding Cdh3 promoter in mouse testis. Moreover, the knockdowns of Junb and/or Fosl2 by siRNA decreased Cdh3 protein levels. Taken together, these data suggest that in 15P-1 Sertoli cells, the AP-1 family members Junb and Fosl2 are responsible for the regulation of Cdh3 expression, which requires the recruitment of both factors to the proximal region of the Cdh3 promoter.

Anomalous Epithelial Variations and Ectopic Inflammatory Response in Chronic Obstructive Pulmonary Disease.

Phenotypic alterations in the lung epithelium have been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but the precise mechanisms orchestrating this persistent inflammatory process remain unknown because of the complexity of lung parenchymal and mesenchymal architecture. To identify cell type-specific mechanisms and cell-cell interactions among the multiple lung resident cell types and inflammatory cells that contribute to COPD progression, we profiled 57,918 cells from lungs of patients with COPD, smokers without COPD, and never-smokers using single-cell RNA sequencing technology. We predicted pseudotime of cell differentiation and cell-to-cell interaction networks in COPD. Although epithelial components in never-smokers were relatively uniform, smoker groups represent extensive heterogeneity in epithelial cells, particularly in alveolar type 2 (AT2) clusters. Among AT2 cells, which are generally regarded as alveolar progenitors, we identified a unique subset that increased in patients with COPD and specifically expressed a series of chemokines including CXCL1 and CXCL8. A trajectory analysis revealed that the inflammatory AT2 cell subpopulation followed a unique differentiation path, and a prediction model of cell-to-cell interactions inferred significantly increased intercellular networks of inflammatory AT2 cells. Our results identify previously unidentified cell subsets and provide an insight into the biological and clinical characteristics of COPD pathogenesis.

Senescent Phenotype of Astrocytes Leads to Activation of BV2 Microglia and N2a Neuronal Cells Death.

(1) Background: Astrocytes, the most abundant cell type in the central nervous system, are essential to tune individual-to-network neuronal activity. Senescence in astrocytes has been discovered as a crucial contributor to several age-related neurological diseases. Here, we aim to observe if astrocytes demonstrate senescence in the process of brain aging, and whether they bring adverse factors, especially harm to neuronal cells. (2) Methods: In vivo, mice were housed for four, 18, and 26 months. An in vitro cell model of aged astrocytes was constructed by serial passaging until passage 20-25, and those within 1-5 were invoked as young astrocytes. Meanwhile, an oxidative induced astrocyte senescence model was constructed by H2O2 induction. (3) Results: In vitro aged astrocytes all showed manifest changes in several established markers of cellular senescence, e.g., P53, P21, and the release of inflammatory cytokine IL-6 and SA-ß-gal positive cells. Results also showed mitochondrial dysfunction in the oxidative stress-induced astrocyte senescence model and treatment of berberine could ameliorate these alterations. Two types of senescent astrocytes' conditioned medium could impact on neuron apoptosis in direct or indirect ways. (4) Conclusions: Senescent astrocyte might affect neurons directly or indirectly acting on the regulation of normal and pathological brain aging.

Notch in Head and Neck Cancer.

Head and neck cancer is a group of neoplastic diseases affecting the facial, oral, and neck region. It is one of the most common cancers worldwide with an aggressive, invasive evolution. Due to the heterogeneity of the tissues affected, it is particularly challenging to study the molecular mechanisms at the basis of these tumors, and to date we are still lacking accurate targets for prevention and therapy. The Notch signaling is involved in a variety of tumorigenic mechanisms, such as regulation of the tumor microenvironment, aberrant intercellular communication, and altered metabolism. Here, we provide an up-to-date review of the role of Notch in head and neck cancer and draw parallels with other types of solid tumors where the Notch pathway plays a crucial role in emergence, maintenance, and progression of the disease. We therefore give a perspective view on the importance of the pathway in neoplastic development in order to define future lines of research and novel therapeutic approaches.

Tumor Environment of Retinoblastoma, Intraocular Cancer.

Retinoblastoma, an intraocular cancer primarily affecting children, interacts with surrounding intraocular and extraocular structures in the development and progression. Subretinal and vitreous seeds are characteristic features of retinoblastoma, which result from the interaction between the tumor and its environment at the levels of tissue and microenvironment. The retina and vitreous affect the disease course and responses to treatment options. Also, neighboring cells in the retina and physicochemical properties of the tumor microenvironment are related to the biological activities of retinoblastoma tumors. Researches focusing on the tumor environment of retinoblastoma will lead to the development of more effective treatment options, which can revolutionize the prognosis of patients with retinoblastoma.

Cancer exosomal microRNAs from gefitinib-resistant lung cancer cells cause therapeutic resistance in gefitinib-sensitive cells.

PURPOSE: Exosomes and their cargo microRNAs play a significant role in various biological processes in cancer. We hypothesized that microRNAs in exosomes secreted by gefitinib-resistant lung cancer cells might induce resistant phenotypes in otherwise gefitinib-sensitive lung cancer cells. METHODS: We isolated exosomes generated by the gefitinib-resistant human lung adenocarcinoma cell line PS-9/ZD. PC-9, which is a gefitinib-sensitive cell line, was treated with the PC-9/ZD exosomes, and these PC-9 cells were analyzed for cell proliferation after treatment with gefitinib. miRNA arrays were analyzed in PC-9 and PC-9/ZD cells, and we isolated microRNAs that were expressed at elevated levels in PC-9/ZD cells. Furthermore, we transfected these microRNAs into PC-9 cells and analyzed the effects on the cells' sensitivity to gefitinib. RESULTS: Exosomes isolated from PC-9/ZD cells significantly increased the proliferation of PC-9 cells during gefitinib treatment. A microRNA array analysis showed that miR-564, miR-658, miR-3652, miR-3126-5p, miR-3682-3p and miR-6810-5p were significantly upregulated in PC-9/ZD cells. PC-9 cells transfected with miR-564 or miR-658 showed chemo-resistant phenotypes. CONCLUSION: Exosomal miR-564 and miR-658 derived from gefitinib-resistant lung cancer cells induce drug resistance in sensitive cells. Cell-to-cell interaction via exosomal microRNAs may be a novel mechanism and therapeutic target of resistance against gefitinib.

IFN-gamma priming of adipose-derived stromal cells at "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2 ) were assessed as alterations of ASCs' CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2 -adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in "wound closure." Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.

Monitoring circadian rhythms of individual cells in plants.

The circadian clock is an endogenous timing system based on the self-sustained oscillation in individual cells. These cellular circadian clocks compose a multicellular circadian system working at respective levels of tissue, organ, plant body. However, how numerous cellular clocks are coordinated within a plant has been unclear. There was little information about behavior of circadian clocks at a single-cell level due to the difficulties in monitoring circadian rhythms of individual cells in an intact plant. We developed a single-cell bioluminescence imaging system using duckweed as the plant material and succeeded in observing behavior of cellular clocks in intact plants for over a week. This imaging technique quantitatively revealed heterogeneous and independent manners of cellular clock behaviors. Furthermore, these quantitative analyses uncovered the local synchronization of cellular circadian rhythms that implied phase-attractive interactions between cellular clocks. The cell-to-cell interaction looked to be too weak to coordinate cellular clocks against their heterogeneity under constant conditions. On the other hand, under light-dark conditions, the heterogeneity of cellular clocks seemed to be corrected by cell-to-cell interactions so that cellular clocks showed a clear spatial pattern of phases at a whole plant level. Thus, it was suggested that the interactions between cellular clocks was an adaptive trait working under day-night cycles to coordinate cellular clocks in a plant body. These findings provide a novel perspective for understanding spatio-temporal architectures in the plant circadian system.

Direct evidence that suspensor cells have embryogenic potential that is suppressed by the embryo proper during normal embryogenesis.

The suspensor is a temporary supporting structure of proembryos. It has been proposed that suspensor cells also possess embryogenic potential, which is suppressed by the embryo as an effect of the embryo-suspensor interaction. However, data to support this hypothesis are not yet available. In this report, using an in vivo living cell laser ablation technique, we show that Arabidopsis suspensor cells can develop into embryos after removing the embryo proper. The embryo proper plays a critical role in maintaining suspensor cell identity. However, this depends on the developmental stage; after the globular embryo stage, the suspensors no longer possess the potential to develop into embryos. We also reveal that hypophysis formation may be essential for embryo differentiation. Furthermore, we show that, after removing the embryo, auxin gradually accumulates in the top suspensor cell where cell division occurs to produce an embryo. Auxin redistribution likely reprograms the fate of the suspensor cell and triggers embryogenesis in suspensor cells. Thus, we provide direct evidence that the embryo suppresses the embryogenic potential of suspensor cells.

Proinflammatory interleukins' production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cell-to-cell interaction.

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL-6 and IL-8 production by adipose-derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O2 concentrations, some highly up-regulated at hypoxia. IL-6 and IL-8 production was inversely dependent on cell culture density. In early (first-third) passages, IL-6 and IL-8 concentration was higher at 20% O2 and in late (8th-12th) passages under 5% O2. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL-6 and did not result in the elevation of IL-8 concentration. Thereby, the production of proinflammatory interleukins (IL-6 and IL-8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood-borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. SIGNIFICANCE PARAGRAPH: Ex vivo expansion is widely used for increasing the number of adipose-derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.

Alteration of PBMC transcriptome profile after interaction with multipotent mesenchymal stromal cells under "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) have demonstrated a pronounced immunosuppressive activity, the manifestation of which depends on the microenvironmental factors, including O2 level. Here we examined the effects of MSCs on transcriptomic profile of allogeneic phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) after interaction at ambient (20%) or "physiological" hypoxia (5%) O2. As revealed with microarray analysis, PBMC transcriptome at 20% O2 was more affected, which was manifested as differential expression of more than 300 genes, whereas under 5% O2 220 genes were changed. Most of genes at 20% O2 were downregulated, while at hypoxia most of genes were upregulated. Altered gene patterns were only partly overlapped at different O2 levels. A set of altered genes at hypoxia only was of particular interest. According to Gene Ontology a part of above genes was responsible for adhesion, cell communication, and immune response. At both oxygen concentrations, MSCs demonstrated effective immunosuppression manifested as attenuation of T cell activation and proliferation as well as anti-inflammatory shift of cytokine profile. Thus, MSC-mediated immunosuppression is executed with greater efficacy at a "physiological" hypoxia, since the same result has been achieved through a change in the expression of a fewer genes in target PBMCs.

The molecular and cellular choreography of early mammalian lung development.

Mammalian lung development starts from a specific cluster of endodermal cells situated within the ventral foregut region. With the orchestrating of delicate choreography of transcription factors, signaling pathways, and cell-cell communications, the endodermal diverticulum extends into the surrounding mesenchyme, and builds the cellular and structural basis of the complex respiratory system. This review provides a comprehensive overview of the current molecular insights of mammalian lung development, with a particular focus on the early stage of lung cell fate differentiation and spatial patterning. Furthermore, we explore the implications of several congenital respiratory diseases and the relevance to early organogenesis. Finally, we summarize the unprecedented knowledge concerning lung cell compositions, regulatory networks as well as the promising prospect for gaining an unbiased understanding of lung development and lung malformations through state-of-the-art single-cell omics.

Roles of Diet-Associated Gut Microbial Metabolites on Brain Health: Cell-to-Cell Interactions between Gut Bacteria and the Central Nervous System.

Gut microbiota have crucial effects on brain function via the gut-brain axis. Growing evidence suggests that this interaction is mediated by signaling molecules derived from dietary components metabolized by the intestinal microbiota. Although recent studies have provided a substantial understanding of the cell-specific effects of gut microbial molecules in gut microbiome-brain research, further validation is needed. This review presents recent findings on gut microbiota-derived dietary metabolites that enter the systemic circulation and influence the cell-to-cell interactions between gut microbes and cells in the central nervous system (CNS), particularly microglia, astrocytes, and neuronal cells, ultimately affecting cognitive function, mood, and behavior. Specifically, this review highlights the roles of metabolites produced by the gut microbiota via dietary component transformation, including short-chain fatty acids, tryptophan metabolites, and bile acid metabolites, in promoting the function and maturation of brain cells and suppressing inflammatory signals in the CNS. We also discuss future directions for gut microbiome-brain research, focusing on diet-induced microbial metabolite-based therapies as possible novel approaches to mental health treatment.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Flagellar Propulsion of Sperm Cells Against a Time-Periodic Interaction Force.

Sperm cells undergo complex interactions with external environments, such as a solid-boundary, fluid flow, as well as other cells before arriving at the fertilization site. The interaction with the oviductal epithelium, as a site of sperm storage, is one type of cell-to-cell interaction that serves as a selection mechanism. Abnormal sperm cells with poor swimming performance, the major cause of male infertility, are filtered out by this selection mechanism. In this study, collinear bundles, consisting of two sperm cells, generate propulsive thrusts along opposite directions and allow to observe the influence of cell-to-cell interaction on flagellar wave-patterns. The developed elasto-hydrodynamic model demonstrates that steric and adhesive forces lead to highly symmetrical wave-pattern and reduce the bending amplitude of the propagating wave. It is measured that the free cells exhibit a mean flagellar curvature of 6.4 ± 3.5 rad mm-1 and a bending amplitude of 13.8 ± 2.8 rad mm-1 . After forming the collinear bundle, the mean flagellar curvature and bending amplitude are decreased to 1.8 ± 1.1 and 9.6 ± 1.4 rad mm-1 , respectively. This study presents consistent theoretical and experimental results important for understanding the adaptive behavior of sperm cells to the external time-periodic force encountered during sperm-egg interaction.

High- and Super-Resolution Imaging of Cell-Cell Interfaces.

Physical interfaces mediate interactions between multiple types of cells. Despite the importance of such interfaces to the cells' function, their high-resolution optical imaging has been typically limited due to poor alignment of the interfaces relative to the optical plane of imaging. Here, we present a simple and robust method to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips and bringing them into contact in a controlled and stable fashion. We demonstrate aberration-free high-resolution imaging of interfaces between live T cells and antigen-presenting cells, known as immune synapses, as an outstanding example. Imaging methods may include multiple diffraction-limited and super-resolution microscopy techniques (e.g., bright-field, confocal, STED, and dSTORM). Thus, our simple and widely compatible approach allows imaging with high- and super-resolution the intricate structure and molecular organization within a variety of cell-cell interfaces.

Self-organized insulin-producing ß-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential.

BACKGROUND: Human omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived ß-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional ß-cells remains challenging. METHODS: We aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing ß-cells via cell-matrix/cell-cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the ß-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes. RESULTS: Our findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing ß-cell progenitors, as evident from the upregulation of pancreatic ß-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated ß-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation. CONCLUSIONS: Our findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic ß-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.

Single-Cell Transcriptomics Revealed Subtype-Specific Tumor Immune Microenvironments in Human Glioblastomas.

Human glioblastoma (GBM), the most aggressive brain tumor, comprises six major subtypes of malignant cells, giving rise to both inter-patient and intra-tumor heterogeneity. The interaction between different tumor subtypes and non-malignant cells to collectively shape a tumor microenvironment has not been systematically characterized. Herein, we sampled the cellular milieu of surgically resected primary tumors from 7 GBM patients using single-cell transcriptome sequencing. A lineage relationship analysis revealed that a neural-progenitor-2-like (NPC2-like) state with high metabolic activity was associated with the tumor cells of origin. Mesenchymal-1-like (MES1-like) and mesenchymal-2-like (MES2-like) tumor cells correlated strongly with immune infiltration and chronic hypoxia niche responses. We identified four subsets of tumor-associated macrophages/microglia (TAMs), among which TAM-1 co-opted both acute and chronic hypoxia-response signatures, implicated in tumor angiogenesis, invasion, and poor prognosis. MES-like GBM cells expressed the highest number of M2-promoting ligands compared to other cellular states while all six states were associated with TAM M2-type polarization and immunosuppression via a set of 10 ligand-receptor signaling pathways. Our results provide new insights into the differential roles of GBM cell subtypes in the tumor immune microenvironment that may be deployed for patient stratification and personalized treatment.

Growth Coordination Between Butyrate-Oxidizing Syntrophs and Hydrogenotrophic Methanogens.

Syntrophy is a thermodynamically required mutualistic cooperation between fatty acid-oxidizing bacteria and methanogens that plays the important role in organic decomposition and methanogenesis in anoxic environments. In this study, three experiments were conducted to evaluate the cell-to-cell interaction in a thermophilic coculture consisting of Syntrophothermus lipocalidus and Methanocella conradii and a mesophilic coculture consisting of Syntrophomonas wolfei and Methanococcus maripaludis. First, syntrophs and methanogens were inoculated at different initial cell ratios to evaluate the growth synchronization. The quantitative PCR analysis revealed that the organism with a lower relative abundance at the beginning always grew faster, and the cell ratio converged over time to relative constant values in both the thermophilic and mesophilic cocultures. Next, intermittent ultrasound and constant shaking treatments were used to evaluate the influence of physical disturbance on microbial aggregation in the mesophilic coculture. The fluorescence in situ hybridization and scanning electron microscopy revealed that the tendency of syntrophic aggregation was not affected by the physical disturbances, although the activity was slightly depressed. Syntrophomonas dominated in the initial microbial aggregates, which, however, did not grow until Methanococcus was attached and increased to a significant extent, indicating the local growth synchronization during the formation and maturation of syntrophic aggregates. Last, microfluidic experiments revealed that whether or not Syntrophomonas or Methanococcus was loaded first, the second organism preferred moving to the place where the first organism was located, suggesting the cell-to-cell attraction between Syntrophomonas and Methanococcus. Collectively, our study demonstrated the growth synchronization and cell-to-cell attraction between the butyrate-oxidizing bacteria and methanogens for optimizing the syntrophic cooperation.