Showing papers on "Cell culture published in 2021"

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Abstract: Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Basic Cell Culture Protocols

Abstract: Culture of Primary Adherent Cells and a Continuously Growing Nonadherent Cell Line Cheryl D. Helgason Detection of Mycoplasma Contaminations Cord C. Uphoff and Hans G. Drexler Eradication of Mycoplasma Contaminations Cord C. Uphoff and Hans G. Drexler Authentication of Scientific Human Cell Lines: Easy-to-Use DNA Fingerprinting Wilhelm G. Dirks and Hans G. Drexler Cytogenetic Analysis of Cell Lines Roderick A. F. MacLeod and Hans G. Drexler Human and Mouse Hematopoietic Colony-Forming Cell Assays Cindy L. Miller and Becky Lai Isolation and Culture of Murine Macrophages John Q. Davies and Siamon Gordon Isolation and Culture of Human Macrophages John Q. Davies and Siamon Gordon Development of T-Lymphocytes in Mouse Fetal Thymus Organ Culture Tomoo Ueno, Cunlan Liu, Takeshi Nitta, and Yousuke Takahama In Vitro Generation of Lymphocytes From Embryonic Stem Cells Renee F. de Pooter, Sarah K. Cho, and Juan Carlos Zuniga-Pflucker Hematopoietic Development of Human Embryonic Stem Cells in Culture Xinghui Tian and Dan S. Kaufman Generation of Murine Stromal Cell Lines: Models for the Microenvironment of the Embryonic Mouse Aorta-Gonads-Mesonephros Region Robert A. J. Oostendorp, Kirsty Harvey, and Elaine A. Dzierzak Culture of Human and Mouse Mesenchymal Cells Emer Clarke Isolation, Purification, and Cultivation of Murine and Human Keratinocytes Frizell L. Vaughan and Ludmila I. Bernstam Isolation and Culture of Primary Human Hepatocytes Edward L. LeCluyse, Eliane Alexandre, Geraldine A. Hamilton, Catherine Viollon-Abadie, D. James Coon, Summer Jolley, and Lysiane Richert Primary Kidney Proximal Tubule Cells Mary Taub Enzymatic Dissociation and Culture of Normal Human Mammary Tissue to Detect Progenitor Activity John Stingl, Joanne T. Emerman, and Connie J. Eaves Generation and Differentiation of Neurospheres From Murine Embryonic Day 14 Central Nervous System Tissue Sharon A. Louis and Brent A. Reynolds Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells Gabi Shefer and Zipora Yablonka-Reuveni Adult Ventricular Cardiomyocytes: Isolation and Culture Klaus-Dieter Schluter and Daniela Schreiber Isolation and Culture of Primary Endothelial Cells Bruno Larrivee and Aly Karsan Studying Leukocyte Rolling and Adhesion In Vitro Under Flow Conditions Susan L. Cuvelier and Kamala D. Patel Isolation and Characterization of Side Population Cells Margaret A. Goodell, Shannon McKinney-Freeman, and Fernando D. Camargo Scalable Production of Embryonic Stem Cell-Derived Cells Stephen M. Dang and Peter W. Zandstra Index

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation.

Abstract: Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

The in vitro antiviral activity of lactoferrin against common human coronaviruses and SARS-CoV-2 is mediated by targeting the heparan sulfate co-receptor.

Abstract: Coronavirus disease 2019 (COVID-19) is an ongoing pandemic that lacks effective therapeutic interventions SARS-CoV-2 infects ACE2-expressing cells and gains cell entry through either direct plasma membrane fusion or endocytosis Recent studies have shown that in addition to ACE2, heparan sulfate proteoglycans (HSPGs) also play an important role in SARS-CoV-2 cell attachment by serving as an attachment factor Binding of viral spike protein to HSPGs leads to the enrichment of local concentration for the subsequent specific binding with ACE2 We therefore hypothesize that blocking the interactions between viral spike protein and the HSPGs will lead to inhibition of viral replication In this study, we report our findings of the broad-spectrum antiviral activity and the mechanism of action of lactoferrin (LF) against multiple common human coronaviruses as well as SARS-CoV-2 Our study has shown that LF has broad-spectrum antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E in cell culture, and bovine lactoferrin (BLF) is more potent than human lactoferrin Mechanistic studies revealed that BLF binds to HSPGs, thereby blocking viral attachment to the host cell The antiviral activity of BLF can be antagonized by the HSPG mimetic heparin Combination therapy experiment showed that the antiviral activity of LF is synergistic with remdesivir in cell culture Molecular modelling suggests that the N-terminal positively charged region in BLF (residues 17-41) confers the binding to HSPGs Overall, LF appears to be a promising drug candidate for COVID-19 that warrants further investigation

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

Abstract: The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.

Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin.

Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2' site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Abstract: Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

T cell circuits that sense antigen density with an ultrasensitive threshold

Abstract: Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic “off-tumor” cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)–T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells—it increases both CAR expression and activation—leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.

Culture of SARS-CoV-2 in a panel of laboratory cell lines, permissivity, and differences in growth profile.

Abstract: The emergence of COVID-19 disease due to SARS-CoV-2 at the end of 2019 was rapidly associated with the isolation of the strain from co-culture onto VERO cells. These isolations quickly made it possible to carry out the first tests for antiviral agents' susceptibility and drug repurposing. However, it seems important to make an inventory of all the cells that can support the growth of this virus and evaluate possible differences between isolates. In the present work, we tested 4 strains of SARS-CoV-2 locally isolated on a panel of 34 cell lines present in our laboratory and commonly used for the isolation of human pathogenic microorganism. After inoculation, cells were observed for cytopathic effects and quantitative real-time polymerase reaction was used to measure the virus replication on the cells. We were able to obtain growth on 7 cell lines, 6 simian, and the human Caco-2. The cytopathogenic effects are variable, ranging from lysis of the cell monolayer in 48-72 h to no cytopathic effect in spite of intense multiplication, as in Caco-2 cells. Interestingly, effect and multiplication varied widely according to the strain tested. In this paper, we explored the species specificity and tissue tropism of SARS-CoV-2 in vitro on a panel of cells available in our laboratory and identified human and animal cell lines susceptible to support SARS-CoV-2 replication. Our work highlights the importance of testing multiple strains when testing antiviral molecules and performing patho-physiological analyzes.

A novel cell culture system modeling the SARS-CoV-2 life cycle.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection.

Abstract: The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.

Abstract: Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2.

Abstract: Influenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan (λ-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses with EC50 values ranging from 0.3 to 1.4 μg/ml, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an EC50 value of 0.9 ± 1.1 μg/ml. No toxicity to the host cells was observed at concentrations up to 300 μg/ml. Plaque titration and western blot analysis verified that λ-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing virus entry. Moreover, its intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, λ-CGN could be a promising antiviral agent for preventing infection with several respiratory viruses.

Cancer cell-derived exosomal circUSP7 induces CD8+ T cell dysfunction and anti-PD1 resistance by regulating the miR-934/SHP2 axis in NSCLC.

Abstract: BACKGROUND CD8+ T cells play a critical role in the innate antitumour immune response. Recently, CD8+ T cell dysfunction has been verified in various malignant cancers, including non-small cell lung cancer (NSCLC). However, the molecular biological mechanisms of CD8+ T cell dysfunction in human NSCLC are still unclear. METHODS The expression of circular ubiquitin-specific protease-7 (circUSP7) in NSCLC tissues, exosomes, and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Exosomes were isolated from the culture medium of NSCLC cells and the plasma of NSCLC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit. The exosomes were then characterized by transmission electronic microscopy (TEM), NanoSight and western blotting. The role of circUSP7 in CD8+ T cell dysfunction was assessed by enzyme-linked immunosorbent assay (ELISA). In vivo circular RNA (circRNA) precipitation (circRIP), RNA immunoprecipitation (RIP), and luciferase reporter assays were performed to explore the molecular mechanisms of circUSP7 in CD8+ T cells. In a retrospective study, the clinical characteristics and prognostic significance of circUSP7 in NSCLC tissues were determined. RESULTS The expression levels of circUSP7 were higher in human NSCLC tissues than in matched adjacent nontumour tissues. Increased levels of circUSP7 indicate poor clinical prognosis and CD8+ T cell dysfunction in patients with NSCLC. The circUSP7 found in NSCLC patient plasma is predominantly secreted by NSCLC cells in an exosomal manner, and circUSP7 inhibits IFN-γ, TNF-α, Granzyme-B and Perforin secretion by CD8+ T cells. Furthermore, circUSP7 inhibits CD8+ T cell function by upregulating the expression of Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) via sponging miR-934. Finally, we show that circUSP7 may promote resistance to anti-PD1 immunotherapy in NSCLC patients. CONCLUSIONS Exosomal circUSP7 is predominantly secreted by NSCLC cells and contributes to immunosuppression by promoting CD8+ T cell dysfunction in NSCLC. CircUSP7 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for NSCLC patients.

Brain immune cells undergo cGAS/STING-dependent apoptosis during herpes simplex virus type 1 infection to limit type I IFN production

Abstract: Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.

Generation of insulin-producing pancreatic β cells from multiple human stem cell lines.

Abstract: We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell-derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6-1+ pancreatic progenitors through the timed application of keratinocyte growth factor, SANT1, TPPB, LDN193189 and retinoic acid. Endocrine induction and subsequent SC-β-cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1 and retinoic acid. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes ~34 d to generate functional SC-β cells, plus an additional 1-2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene. This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology laboratories. In addition to expanding protocol accessibility and simplifying SC-β-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.

Resibufogenin inhibited colorectal cancer cell growth and tumorigenesis through triggering ferroptosis and ROS production mediated by GPX4 inactivation.

Abstract: Resibufogenin (RB) has been used for cancer treatment, but the underlying mechanisms are still unclear. This study aimed to investigate the effects of RB treatment on colorectal cancer (CRC) cells, and to determine the underlying mechanisms. The cell counting kit-8 assay was used to determine cell viability. Cell morphology was observed under light microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to detect cell apoptosis. Intracellular ferrous iron (Fe2+ ), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species levels were detected by using commercial iron assay kit, MDA assay kit, GSH assay kit, and 2,7-dichlorodihydrofluorescein diacetate probes, respectively. The protein expressions were determined by Western blot and immunohistochemistry. RB inhibited cell viability in the CRC cell lines (HT29 and SW480) in a dose- and time-dependent manner, and caused cytotoxicity to the normal colonic epithelial cell line (NCM460) at high dose. Similarly, RB induced morphological changes in CRC cells from normal to round shape, and promoted cell death. Of note, RB triggered oxidative stress and ferroptotic cell death in CRC cells, and only ferroptosis inhibitors (deferoxamine and ferrostatin-1), instead of inhibitors for other types of cell death (apoptosis, autophagy, and necroptosis), reversed the inhibitory effects of RB on CRC cell proliferation. Furthermore, glutathione peroxidase 4 (GPX4) was inactivated by RB treatment, and overexpression of GPX4 alleviated RB-induced oxidative cell death in CRC cells. Consistently, the in vivo experiments validated that RB also triggered oxidative stress, and inhibited CRC cells growth and tumorigenicity in mice models. RB can inhibit CRC cells growth and tumorigenesis by triggering ferroptotic cell death in a GPX4 inactivation-dependent manner.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Abstract: Background: Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. Although Calu-3, a human lung cell line which endogenously expresses ACE2, supports SARS-CoV-2 replication, they are significantly less permissive to infection than other models. Furthermore, ACE2 expression in the respiratory tract is low and emerging evidence suggests the utilization of alternative host cell receptors and attachment factors may compensate for low ACE2 expression levels in the lung. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Methods: A panel of 10 cell lines, with variable expression levels of ACE2 and TMPRSS2 were infected with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020. Viral replication was monitored through assessment of cell-associated and cell-free viral RNA (vRNA) by QRT-PCR as well as N staining by FACS and in situ hybridization. Effect of blocking S protein by neutralizing antibodies and an ACE2-Fc decoy peptide, ACE2 blocking by a specific antibody, and ACE2 knockout by CRISPR on SARS-CoV-2 replication was determined by Q-RT-PCR for vRNAs. Various viral entry inhibitors were used to pathway of SARS-CoV-2 entry in H522 cells. RNA sequencing and proteomics was used to study the cell and innate immune responses in infected H522 cells. siRNA-mediated knockdown was utilized to further characterize the pathway of immune sensing. Results: Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5- dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. Conclusion: These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Quercetin induces pro-apoptotic autophagy via SIRT1/AMPK signaling pathway in human lung cancer cell lines A549 and H1299 in vitro

Abstract: Background Quercetin, a natural flavonoid compound, is a potent cancer therapeutic agent widely found in fruit and vegetables. It has been reported to induce growth inhibition and apoptosis in both A549 and H1299 human lung cancer cells. However, the effect of quercetin-induced autophagy on apoptosis and the possible autophagy mechanism in A549 and H1299 cells have not yet been critically examined. Methods A549 and H1299 cells were treated with different concentrations of quercetin for 24 hours. Cell growth was measured by cell counting kit-8 (CCK-8) assay, whereas apoptosis was assessed by western blotting analysis of apoptotic proteins. The levels of proteins and genes involved in autophagy were determined by western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Autophagosomes were also observed by transmission electron microscopy (TEM) and LC3 immunofluorescence. Results Quercetin inhibited cell viability and induced mitochondria-dependent apoptosis in both A549 and H1299 cells in a dose-dependent. Moreover, quercetin also promoted the expression of LC3-II and beclin 1 and suppressed the expression of p62. The mRNA levels of LC3-II, beclin 1, Atg5, Atg7, and Atg12 were upregulated by quercetin treatment. Autophagy inhibition with 3-methyladenine could effectively inhibit quercetin-induced apoptosis. In addition, quercetin dose-dependently elevated the levels of SIRT1 protein and the pAMPK-AMPK ratio. Quercetin-induced autophagy was attenuated by SIRT1 inhibitor EX527 and SirT1 knockdown by small interfering RNA (siRNA). Conclusions Quercetin-induced autophagy contributes to apoptosis in A549 and H1299 lung cancer cells, which involved the SIRT1/AMPK signaling pathway.

Muscle stem cell isolation and in vitro culture for meat production: A methodological review.

Abstract: Cultured muscle tissue-based protein products, also known as cultured meat, are produced through in vitro myogenesis involving muscle stem cell culture and differentiation, and mature muscle cell processing for flavor and texture. This review focuses on the in vitro myogenesis for cultured meat production. The muscle stem cell-based in vitro muscle tissue production consists of a sequential process: (1) muscle sampling for stem cell collection, (2) muscle tissue dissociation and muscle stem cell isolation, (3) primary cell culture, (4) upscaled cell culture, (5) muscle differentiation and maturation, and (6) muscle tissue harvest. Although muscle stem cell research is a well-established field, the majority of these steps remain to be underoptimized to enable the in vitro creation of edible muscle-derived meat products. The profound understanding of the process would help not only cultured meat production but also business sectors that have been seeking new biomaterials for the food industry. In this review, we discuss comprehensively and in detail each step of cutting-edge methods for cultured meat production. This would be meaningful for both academia and industry to prepare for the new era of cellular agriculture.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Abstract: Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro.

Abstract: Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

In vitro and ex vivo models of adipocytes.

Abstract: Adipocytes are specialized cells with pleiotropic roles in physiology and pathology. Several types of fat cells with distinct metabolic properties coexist in various anatomically defined fat depots in mammals. White, beige, and brown adipocytes differ in their handling of lipids and thermogenic capacity, promoting differences in size and morphology. Moreover, adipocytes release lipids and proteins with paracrine and endocrine functions. The intrinsic properties of adipocytes pose specific challenges in culture. Mature adipocytes float in suspension culture due to high triacylglycerol content and are fragile. Moreover, a fully differentiated state, notably acquirement of the unilocular lipid droplet of white adipocyte, has so far not been reached in two-dimensional culture. Cultures of mouse and human-differentiated preadipocyte cell lines and primary cells have been established to mimic white, beige, and brown adipocytes. Here, we survey various models of differentiated preadipocyte cells and primary mature adipocyte survival describing main characteristics, culture conditions, advantages, and limitations. An important development is the advent of three-dimensional culture, notably of adipose spheroids that recapitulate in vivo adipocyte function and morphology in fat depots. Challenges for the future include isolation and culture of adipose-derived stem cells from different anatomic location in animal models and humans differing in sex, age, fat mass, and pathophysiological conditions. Further understanding of fat cell physiology and dysfunction will be achieved through genetic manipulation, notably CRISPR-mediated gene editing. Capturing adipocyte heterogeneity at the single-cell level within a single fat depot will be key to understanding diversities in cardiometabolic parameters among lean and obese individuals.

SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells.

Abstract: Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated β-galactosidase (SA-β-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Abstract: Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

IFNγ-mediated repression of system xc- drives vulnerability to induced ferroptosis in hepatocellular carcinoma cells.

Abstract: IFNγ released from CD8+ T cells or natural killer cells plays a crucial role in antitumor host immunity. Several studies have found that IFNγ is involved in regulating tumor cell proliferation and apoptosis. However, few studies have examined its role in cell ferroptosis. Here, we found that IFNγ treatment enhanced glutathione depletion, promoted cell cycle arrested in G0/G1 phase, increased lipid peroxidation, and sensitized cells to ferroptosis activators. Additionally, IFNγ down-regulated the mRNA and protein levels of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc- via activating the JAK/STAT pathway in hepatocellular carcinoma (HCC) cell lines. Furthermore, IFNγ increased reactive oxygen species levels and decreased mitochondiral membrane potential in Bel7402 and HepG2 cells. These changes were accompanied by decreased system xc- activity. Cancer cells exposed to TGFβ1 for 48 h showed sensitization to IFNγ + erastin-induced ferroptosis, with decreased system xc- expression. In conclusion, IFNγ repressed system xc- activation via activating JAK/STAT signaling. Additionally, enhanced lipid peroxidation was associated with altered mitochondrial function in HCC cells. Our findings identified a role for IFNγ in sensitizing HCC cells to ferroptosis, which provided new insights for applying IFNγ as a cancer treatment.

Rapid reprogramming of tumour cells into cancer stem cells on double-network hydrogels.

Abstract: Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells. A double-network hydrogel can rapidly reprogramme differentiated tumour cells into cancer stem cells that display elevated tumorigenicity in vivo.