Cyclic uniaxial mechanical stretching of cells using a LEGO® parts-based mechanical stretcher system.

Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO® parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.

Interferon regulatory factor 4 sustains CD8(+) T cell expansion and effector differentiation.

Upon infection, CD8(+) T cells undergo a stepwise process of early activation, expansion, and differentiation into effector cells. How these phases are transcriptionally regulated is incompletely defined. Here, we report that interferon regulatory factor 4 (IRF4), dispensable for early CD8(+) T cell activation, was vital for sustaining the expansion and effector differentiation of CD8(+) T cells. Mechanistically, IRF4 promoted the expression and function of Blimp1 and T-bet, two transcription factors required for CD8(+) T cell effector differentiation, and simultaneously repressed genes that mediate cell cycle arrest and apoptosis. Selective ablation of Irf4 in peripheral CD8(+) T cells impaired antiviral CD8(+) T cell responses, viral clearance, and CD8(+) T cell-mediated host recovery from influenza infection. IRF4 expression was regulated by T cell receptor (TCR) signaling strength via mammalian target of rapamycin (mTOR). Our data reveal that IRF4 translates differential strength of TCR signaling into different quantitative and qualitative CD8(+) T cell responses.

The polycomb protein Ezh2 regulates differentiation and plasticity of CD4(+) T helper type 1 and type 2 cells.

After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.

Tau modulates Schwann cell proliferation, migration and differentiation following peripheral nerve injury.

Tau protein (encoded by the gene microtubule-associated protein tau, Mapt) is essential for the assembly and stability of microtubule and the functional maintenance of the nervous system. Tau is highly abundant in neurons and is detectable in astrocytes and oligodendrocytes. However, whether tau is present in Schwann cells, the unique glial cells in the peripheral nervous system, is unclear. Here, we investigated the presence of tau and its coding mRNA, Mapt, in cultured Schwann cells and find that tau is present in these cells. Gene silencing of Mapt promoted Schwann cell proliferation and inhibited Schwann cell migration and differentiation. In vivo application of Mapt siRNA suppressed the migration of Schwann cells after sciatic nerve injury. Consistent with this, Mapt-knockout mice showed elevated proliferation and reduced migration of Schwann cells. Rats injected with Mapt siRNA and Mapt-knockout mice also exhibited impaired myelin and lipid debris clearance. The expression and distribution of the cytoskeleton proteins α-tubulin and F-actin were also disrupted in these animals. These findings demonstrate the existence and biological effects of tau in Schwann cells, and expand our understanding of the function of tau in the nervous system.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Involvement of Progranulin and Granulin Expression in Inflammatory Responses after Cerebral Ischemia.

Progranulin (PGRN) plays a crucial role in diverse biological processes, including cell proliferation and embryonic development. PGRN can be cleaved by neutrophil elastase to release granulin (GRN). PGRN has been found to inhibit inflammation. Whereas, GRN plays a role as a pro-inflammatory factor. However, the pathophysiological roles of PGRN and GRN, at early stages after cerebral ischemia, have not yet been fully understood. The aim of this study was to obtain further insight into the pathologic roles of PGRN and GRN. We demonstrated that the amount of PGRN was significantly increased in microglial cells after cerebral ischemia in rats and that neutrophil elastase activity was also increased at an early stage after cerebral ischemia, resulting in the production of GRN. The inhibition of neutrophil elastase activity suppressed PGRN cleavage and GRN production, as well as the increase in pro-inflammatory cytokines, after cerebral ischemia. The administration of an elastase inhibitor decreased the number of injured cells and improved the neurological deficits test scores. Our findings suggest that an increase in the activity of elastase to cleave PGRN, and to produce GRN, was involved in an inflammatory response at the early stages after cerebral ischemia, and that inhibition of elastase activity could suppress the progression of cerebral ischemic injury.

Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78).

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C > T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with fronto-temporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

Directing adipose-derived stem cells into keratinocyte-like cells: impact of medium composition and culture condition.

BACKGROUND: Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine to restore the epidermal barrier. OBJECTIVE: This study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. In particular, the impact of different culture conditions (media compositions, 2D, 3D cultures) and extracellular matrix (ECM) molecules was evaluated. METHODS: Adipose-derived stem cells derived from subcutaneous abdominal fat were characterized by stemness-associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. To evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR. RESULTS: We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced. CONCLUSION: Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures.

Cell fixation and preservation for droplet-based single-cell transcriptomics.

BACKGROUND: Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. METHODS: Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. RESULTS: By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. CONCLUSIONS: We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.

Establishment and characterization of a canine keratinocyte organoid culture system.

BACKGROUND: Perturbations of epidermal and follicular homeostasis have been attributed to a variety of skin diseases affecting dogs. The availability of an in vitro system to investigate these diseases is important to understand underlying pathomechanisms. OBJECTIVES: To establish an accurate and reliable in vitro 3D system of canine keratinocyte organoids to lay the basis for studying functional defects in interfollicular epidermis (IFE) and hair follicle (HF) morphogenesis, reconstitution and differentiation that lead to alopecic and epidermal diseases. ANIMALS: Skin biopsies were obtained from freshly euthanized dogs of different breeds with no skin abnormalities. METHODS: Cells derived from microdissected IFE and HFs were seeded in Matrigel and keratinocyte organoids were grown and characterized using immunohistochemistry, RT-qPCR and RNA sequencing. RESULTS: Both organoid lines develop into a basal IFE-like cell type. Gene and protein expression analysis revealed high mRNA and protein levels of keratins 5 and 14, IFE differentiation markers and intercellular molecules. Key markers of HF stem cells were lacking. Withdrawal of growth factors resulted in upregulation of markers such as KRT16, Involucrin, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. CONCLUSION AND CLINICAL IMPORTANCE: Our 3D in vitro culture system provides the basis to explore epidermal function, to investigate the culture conditions necessary for the development of organoids with a HF signature and to address cutaneous disorders in dogs. However, for induction of HF signatures or hair growth, addition of different growth factors or co-culture with dermal papilla will be required.

Fundamentals of diffusion MRI physics.

Diffusion MRI is commonly considered the "engine" for probing the cellular structure of living biological tissues. The difficulty of this task is threefold. First, in structurally heterogeneous media, diffusion is related to structure in quite a complicated way. The challenge of finding diffusion metrics for a given structure is equivalent to other problems in physics that have been known for over a century. Second, in most cases the MRI signal is related to diffusion in an indirect way dependent on the measurement technique used. Third, finding the cellular structure given the MRI signal is an ill-posed inverse problem. This paper reviews well-established knowledge that forms the basis for responding to the first two challenges. The inverse problem is briefly discussed and the reader is warned about a number of pitfalls on the way.

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling.

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors.

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.

Geometry of the Gene Expression Space of Individual Cells.

There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the vertices represent specialists at key tasks.

A multi-center analysis of adverse events among two thousand, three hundred and seventy two adult patients undergoing adult autologous stem cell therapy for orthopaedic conditions.

INTRODUCTION: The purpose of the present investigation is to report on detailed complications among a much larger group of 2372 orthopaedic patients treated with stem cell injections who were followed in a treatment registry for up to nine years. METHODS: All patients underwent an MSC-based, percutaneous injection treatment of an orthopaedic condition between December 2005 and September 2014 at one of 18 clinical facilities. Treated areas of the body included the knee, hip, ankle/foot, hand/wrist, elbow, shoulder, and spine. The patients were followed prospectively via enrollment in a treatment registry. Patients were followed prospectively at one, three, six and 12 months, and annually thereafter, using an electronic system, ClinCapture software. RESULTS: A total of 3012 procedures were performed on 2372 patients with follow-up period of 2.2 years. A total of 325 adverse events were reported. The majority were pain post-procedure (n = 93, 3.9 % of the study population) and pain due to progressive degenerative joint disease (n = 90, 3.8 % of the study population). Seven cases reported neoplasms, a lower rate than in the general population. The lowest rate of adverse events was observed among patients injected with BMC alone. CONCLUSION: Lowest rate of adverse events was among those patients receiving BMC injections alone, but the higher rate of AEs for BMC plus adipose and cultured cells was readily explained by the nature of the therapy or the longer follow-up. There was no clinical evidence to suggest that treatment with MSCs of any type in this study increased the risk of neoplasm.

Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity.

In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo.

AngiomiR-126 expression and secretion from circulating CD34(+) and CD14(+) PBMCs: role for proangiogenic effects and alterations in type 2 diabetics.

Several peripheral blood mononuclear cell (PBMC)-derived cell populations can promote angiogenesis, and differences in CD34(+) or CD14(+) surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34(+)/CD14(+), CD34(+)/CD14(-), CD34(-)/CD14(+), and CD34(-)/CD14(-) PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34(+)/14(+) and CD34(+)/14(-) PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34(-) PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34(+) compared with CD34(-) PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti-miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34(+) PBMCs. MiR-126 levels in supernatants of CD34(+) PBMC subsets were substantially higher compared with CD34(-) PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34(+) PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34(+) PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34(+) PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34(+) PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes.

The primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCR(hi)CD5(int)CD69(-) double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8(int)PD-L1(hi)CD44(hi)EOMES(+) innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

New and TALENted genome engineering toolbox.

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.