Showing papers on "Cell culture published in 2011"
TL;DR: This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins, and hypothesize that ext racellular miRNA are in the most part by-products of dead cells that remain in extrace cellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex.
Abstract: MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.
1,712 citations
TL;DR: It is demonstrated that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle, and is probably required for HIV-1 restriction.
Abstract: The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutieres syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.
1,347 citations
TL;DR: A cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined is formed, and improved derivation efficiencies of vector-free human iPSCs with an episomal approach are demonstrated.
Abstract: We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
1,330 citations
TL;DR: It is proposed that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson’s disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α- Synuclein pathogenicity.
Abstract: Pathogenic aggregation of α-synuclein (αSyn) is implicated in Parkinson's disease and related disorders. αSyn has long been regarded as a natively unfolded monomer that acquires secondary structure only when it binds to its target. This model is the basis for a number of published studies, but Bartels et al. report that endogenous αSyn isolated under entirely non-denaturing conditions from brain tissue, cell lines and living human cells occurs principally as a folded tetramer. This finding suggests that destabilization of the αSyn tetramer precedes misfolding and aggregation in synucleinopathies, and that agents that stabilize the normal tetramer may reduce αSyn pathogenicity. Parkinson’s disease is the second most common neurodegenerative disorder1,2. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson’s disease3,4. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites5, the cytopathological hallmarks of Parkinson’s disease and related disorders called synucleinopathies4. α-Synuclein has long been defined as a ‘natively unfolded’ monomer of about 14 kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles7. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58 kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson’s disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.
1,090 citations
TL;DR: It is shown that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH).
972 citations
TL;DR: The generation of antitumor CD8+ T cell responses requires type I interferon responsiveness in host antigen-presenting cells and the response is dominated by T-cells that secrete polypeptide A into the T cells of the immune system.
Abstract: Despite lack of tumor control in many models, spontaneous T cell priming occurs frequently in response to a growing tumor. However, the innate immune mechanisms that promote natural antitumor T cell responses are undefined. In human metastatic melanoma, there was a correlation between a type I interferon (IFN) transcriptional profile and T cell markers in metastatic tumor tissue. In mice, IFN-β was produced by CD11c+ cells after tumor implantation, and tumor-induced T cell priming was defective in mice lacking IFN-α/βR or Stat1. IFN signaling was required in the hematopoietic compartment at the level of host antigen-presenting cells, and selectively for intratumoral accumulation of CD8α+ dendritic cells, which were demonstrated to be essential using Batf3−/− mice. Thus, host type I IFNs are critical for the innate immune recognition of a growing tumor through signaling on CD8α+ DCs.
929 citations
TL;DR: In vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model, providing compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.
887 citations
TL;DR: It is found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined, and a tumor-suppressive function of NF-σB that contributes to the outcome of cancer therapy is identified.
Abstract: Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
729 citations
TL;DR: It is shown that AhR is required for the postnatal expansion of intestinal RORγt+ ILC and the formation of intestinal lymphoid follicles, establishing a molecular link between nutrients and theformation of immune system components required to maintain intestinal homeostasis and resistance to infections.
Abstract: Innate lymphoid cells (ILC) expressing the transcription factor RORγt induce the postnatal formation of intestinal lymphoid follicles and regulate intestinal homeostasis. RORγt(+) ILC express the aryl hydrocarbon receptor (AhR), a highly conserved, ligand-inducible transcription factor believed to control adaptation of multicellular organisms to environmental challenges. We show that AhR is required for the postnatal expansion of intestinal RORγt(+) ILC and the formation of intestinal lymphoid follicles. AhR activity within RORγt(+) ILC could be induced by dietary ligands such as those contained in vegetables of the family Brassicaceae. AhR-deficient mice were highly susceptible to infection with Citrobacter rodentium, a mouse model for attaching and effacing infections. Our results establish a molecular link between nutrients and the formation of immune system components required to maintain intestinal homeostasis and resistance to infections.
708 citations
TL;DR: It is shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche, which is believed to be the first evidence that the H SC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases.
Abstract: HSC homing, quiescence, and self-renewal depend on the bone marrow HSC niche A large proportion of solid tumor metastases are bone metastases, known to usurp HSC homing pathways to establish footholds in the bone marrow However, it is not clear whether tumors target the HSC niche during metastasis Here we have shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche Importantly, increasing the niche size promoted metastasis, whereas decreasing the niche size compromised dissemination Furthermore, disseminated PCa cells could be mobilized out of the niche and back into the circulation using HSC mobilization protocols Finally, once in the niche, tumor cells reduced HSC numbers by driving their terminal differentiation These data provide what we believe to be the first evidence that the HSC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases Our work may lead to better understanding of the molecular events involved in bone metastases and new therapeutic avenues for an incurable disease
624 citations
TL;DR: increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC, which has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells.
TL;DR: The data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.
Abstract: Tumor cell lines are often used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of ∼100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized ∼4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.
TL;DR: A robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue is described, which represents a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes.
Abstract: Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.
University of Sheffield1, Newcastle upon Tyne Hospitals NHS Foundation Trust2, Agency for Science, Technology and Research3, Royan Institute4, Stanford University5, Boston Children's Hospital6, University of Nottingham7, University of Southern California8, Hebrew University of Jerusalem9, Tel Aviv Sourasky Medical Center10, Cedars-Sinai Medical Center11, University of Geneva12, Manchester Academic Health Science Centre13, University of Manchester14, Genome Institute of Singapore15, Seoul National University16, Harvard University17, University of Edinburgh18, Masaryk University19, WiCell20, University of São Paulo21, Central South University22, University College London23, Karolinska Institutet24, Jawaharlal Nehru Centre for Advanced Scientific Research25, Kyoto University26, Shanghai Jiao Tong University27, Kurchatov Institute28, Russian Academy of Sciences29, Vrije Universiteit Brussel30, King's College London31, Leiden University32, University of Helsinki33, Yale University34, Hospital for Sick Children35, University of New South Wales36, University of Tampere37, Commonwealth Scientific and Industrial Research Organisation38
TL;DR: Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells, and single-nucleotide polymorphism analysis revealed that they included representatives of most major ethnic groups.
Abstract: The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.
TL;DR: It is observed that Au nanorods have distinct effects on cell viability via killing cancer cells while posing negligible impact on normal cells and mesenchymal stem cells, which provides guidance for the design of organelle-targeted nanomaterials in tumor therapy.
Abstract: We have observed that Au nanorods (NRs) have distinct effects on cell viability via killing cancer cells while posing negligible impact on normal cells and mesenchymal stem cells. Obvious differences in cellular uptake, intracellular trafficking, and susceptibility of lysosome to Au NRs by different types of cells resulted in selective accumulation of Au NRs in the mitochondria of cancer cells. Their long-term retention decreased mitochondrial membrane potential and increased reactive oxygen species level that enhances the likelihood of cell death. These findings thus provide guidance for the design of organelle-targeted nanomaterials in tumor therapy.
TL;DR: The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application and provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.
Abstract: Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.
TL;DR: Efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells is reported, indicating functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.
Abstract: The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke’s pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions. Mouse embryonic stem cells are induced to form functional anterior pituitary tissue in three-dimensional culture. A three-dimensional cell culture system has been developed that produces functioning pituitary tissue from mouse embryonic stem cells. The cells differentiate into layered structures, the development of which depends on close juxtaposition of two tissue types in formations that resemble local tissue reactions seen in vivo. Endocrine cells including corticotrophs and somatotrophs are produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone, and transplantation of the pituitary tissues into a hypopituitary mouse model rescued a lethal deficiency in adrenocorticotropin. This work opens the possibility of developing regenerative therapies for pituitary defects, a major category of endocrinological disorders that includes empty sella syndrome, Sheehan syndrome and pituitary apoplexy.
TL;DR: Data from experiments investigating the effects of cell concentration, interstitial flow rate, receptor activity, and focal adhesion kinase phosphorylation support the hypothesis that the competing stimulus is integrin mediated, and provide further evidence that CCR7-dependent autologous chemotaxis is the mechanism that leads to migration with the flow.
Abstract: Interstitial flow is the convective transport of fluid through tissue extracellular matrix. This creeping fluid flow has been shown to affect the morphology and migration of cells such as fibroblasts, cancer cells, endothelial cells, and mesenchymal stem cells. A microfluidic cell culture system was designed to apply stable pressure gradients and fluid flow and allow direct visualization of transient responses of cells seeded in a 3D collagen type I scaffold. We used this system to examine the effects of interstitial flow on cancer cell morphology and migration and to extend previous studies showing that interstitial flow increases the metastatic potential of MDA-MB-435S melanoma cells [Shields J, et al. (2007) Cancer Cell 11:526-538]. Using a breast carcinoma line (MDA-MB-231) we also observed cell migration along streamlines in the presence of flow; however, we further demonstrated that the strength of the flow as well as the cell density determined directional bias of migration along the streamline. In particular, we found that cells either at high seeding density or with the CCR-7 receptor inhibited migration against, rather than with the flow. We provide further evidence that CCR7-dependent autologous chemotaxis is the mechanism that leads to migration with the flow, but also demonstrate a competing CCR7-independent mechanism that causes migration against the flow. Data from experiments investigating the effects of cell concentration, interstitial flow rate, receptor activity, and focal adhesion kinase phosphorylation support our hypothesis that the competing stimulus is integrin mediated. This mechanism may play an important role in development of metastatic disease.
TL;DR: It is demonstrated that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon and several strains of measles virus were shown to use PVRL4 as a receptor.
Abstract: Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors Of these, only the human tumor cell marker PVRL4 (Nectin 4) rendered cells amenable to measles virus infections Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell Several strains of measles virus were also shown to use PVRL4 as a receptor Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a property that is characteristic of receptor-associated viral infections
TL;DR: Methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart are described and general principles for the beginner are outlined, but also detailed specific protocols and advice for common caveats are provided.
TL;DR: An implanted device using blue-light–triggered expression of the glucagon-like peptide 1 attenuates diabetes in mice using a synthetic signaling cascade enabling light-inducible transgene expression in different cell lines grown in culture or bioreactors or implanted into mice.
Abstract: Synthetic biology has advanced the design of genetic devices that can be used to reprogram metabolic activities in mammalian cells. By functionally linking the signal transduction of melanopsin to the control circuit of the nuclear factor of activated T cells, we have designed a synthetic signaling cascade enabling light-inducible transgene expression in different cell lines grown in culture or bioreactors or implanted into mice. In animals harboring intraperitoneal hollow-fiber or subcutaneous implants containing light-inducible transgenic cells, the serum levels of the human glycoprotein secreted alkaline phosphatase could be remote-controlled with fiber optics or transdermally regulated through direct illumination. Light-controlled expression of the glucagon-like peptide 1 was able to attenuate glycemic excursions in type II diabetic mice. Synthetic light-pulse-transcription converters may have applications in therapeutics and protein expression technology.
TL;DR: The pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.
Abstract: Recent evidence has unveiled the critical role of tumor cells with stem cell activities in tumorigenicity and drug resistance, but how tumor microenvironments regulate cancer stem/initiating cells (CSCs) remains unknown. We clarified the role of tumor-associated macrophages (TAMs) and their downstream factor milk-fat globule-epidermal growth factor-VIII (MFG-E8) in the regulation of CSC activities. Bone marrow chimeric systems and adoptive cell transfers elucidated the importance of MFG-E8 from TAMs in conferring to CSCs with the ability to promote tumorigenicity and anticancer drug resistance. MFG-E8 mainly activates signal transducer and activator of transcription-3 (Stat3) and Sonic Hedgehog pathways in CSCs and further amplifies their anticancer drug resistance in cooperation with IL-6. Thus, the pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.
TL;DR: A simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which Cells are in direct contact with each other and with extracellular matrix components is described.
Abstract: Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.
TL;DR: The results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry and may play a protective role in host defense against SARS -CoV infection through binding to HSPG and blocking the preliminary interaction between Sars- coV and host cells.
Abstract: It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.
TL;DR: CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma and it was found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells.
Abstract: CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8(+) T cells and associated with an increased frequency of antigen-specific CD8(+) T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)-depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells.
TL;DR: A high-throughput size-based separation method for processing diluted blood using inertial microfluidics is introduced, demonstrating the isolation of cancer cells spiked in blood by exploiting the difference in size between CTCs and hematologic cells.
Abstract: Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ∼10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.
TL;DR: It is concluded that TMEM16A carries nearly all CaCCCurrent current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia, and potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.
TL;DR: It is reported that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs and shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from h ESCs by specifically regulating Noggin and retinoids signals.
Abstract: Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.
TL;DR: Treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters associated with MDR, as well as epithelial–mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells.
Abstract: Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial–mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance.
TL;DR: Results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells.