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Ependymomas are rare central nervous system tumours that can arise in the brain’s supratentorial region or posterior fossa, or in the spinal cord. In 1924, Percival Bailey published the first comprehensive study of ependymomas. Since then, and especially over the past 10 years, our understanding of ependymomas has grown exponentially. In this Review, we discuss the evolution in knowledge regarding ependymoma subgroups and the resultant clinical implications. We also discuss key oncogenic and tumour suppressor signalling pathways that regulate tumour growth, the role of epigenetic dysregulation in the biology of ependymomas, and the various biological features of ependymoma tumorigenesis, including cell immortalization, stem cell-like properties, the tumour microenvironment and metastasis. We further review the limitations of current therapies such as relapse, radiation-induced cognitive deficits and chemotherapy resistance. Finally, we highlight next-generation therapies that are actively being explored, including tyrosine kinase inhibitors, telomerase inhibitors, anti-angiogenesis agents and immunotherapy.Our understanding of ependymomas, which are rare tumours of the central nervous system, has increased substantially over the past 10 years. This Review discusses important biological features of ependymoma as well as key oncogenes, tumour suppressors and epigenetic changes that could potentially be exploited to improve therapy.

The study of cells aids in comprehending the pathophysiology of diseases. However, obtaining a large number of primary cells in a short period is challenging, and they senesce and die after repeated passages. Therefore, establishing immortalized cell lines is necessary for conducting cellular experiments. Researchers commonly use antibiotics to screen immortalized cell models upon construction. However, due to the low transfection rate of the immortalized genes, a significant number of nonimmortalized cells are killed. The connections between the cells act as a web that floats when many cells die. As a result, successfully transfected immortalized cells are lifted and carried away, leading to only a small number of immortalized cells surviving. The surviving cells survive in the absence of other cell‐secreted factors. However, their proliferative ability is limited, which makes obtaining immortalized cell lines a time‐consuming process. This study aimed to shorten the time required to obtain immortalized cell lines by constructing immortalized Schwann cells and improving the traditional screening method. The immortalized gene transfectants were first cultured, passaged, and then screened. A comparison with the traditional screening method demonstrated the feasibility and advantages of the improved method.

Multiple viruses that are highly pathogenic in humans are known to have evolved in bats. How bats tolerate infection with these viruses, however, is poorly understood. As viruses engage in a wide range of interactions with their hosts, it is essential to study bat viruses in a system that resembles their natural environment like bat-derived in vitro cellular models. However, stable and accessible bat cell lines are not widely available for the broader scientific community. Here, we generated in vitro reagents for the Seba’s short-tailed bat ( Carollia perspicillata ), tested multiple methods of immortalization, and characterized their susceptibility to virus infection and response to immune stimulation. Using pseudotyped virus library and authentic virus infections, we show that these C. perspicillata cell lines derived from a diverse array of tissues are susceptible to viruses bearing the glycoprotein of numerous orthohantaviruses, including Andes and Hantaan virus and are also susceptible to live hantavirus infection. Furthermore, stimulation with synthetic double-stranded RNA prior to infection with vesicular stomatitis virus and Middle Eastern respiratory syndrome coronavirus induced a protective antiviral response, demonstrating the suitability of our cell lines to study the bat antiviral immune response. Taken together, the approaches outlined here will inform future efforts to develop in vitro tools for virology from non-model organisms and these C. perspicillata cell lines will enable studies on virus–host interactions in these bats.

Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15 INK4B , p14 ARF and p16 INK4A ), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15 INK4B is markedly stronger than p16 INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15 INK4B , but not those expressing HRas and lacking p16 INK4A , develop early-onset bladder tumors. The potency of CDKN2B/p15 INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15 INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15 INK4B on cell proliferation, and unveil mechanisms whereby p15 INK4B aberrations may underpin cancer and non-cancer conditions. The human chromosome locus 9p21.3 is a tumour suppressor hub which encodes three CDK inhibitors, p15INK4B, p14ARF and p16INK4A. Here, the authors show that p15INK4B inhibits the cell cycle and glycolysis in a murine model of HRas + ‐mediated urothelial carcinoma and has a more relevant role as a tumour suppressor than its neighbouring p16INK4A.

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing, B cell receptors (BCR) repertoire profiling, CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment, polyreactive, and derived from the natural repertoire. Furthermore, B cells with aPL specificities are not eliminated in patients with PAPS, persist until the memory and long-lived plasma cell stages, likely after defective germinal center selection, while becoming less polyreactive. Lastly, compared with the non-PAPS cells, PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS. Primary antiphospholipid syndrome (PAPS) is a clotting disorder attributed to autoreactive antibodies produced by B cells. Here the authors show, using single cell omics and B cell repertoire data, that autoreactive B cells originate from the natural B cell repertoire and escape germinal center selection to persist in PAPS patient via potential dysregulation of mTORC1 and MYC pathways.

Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.

Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

Newcastle disease virus (NDV), as an avian pathogen, can infect a broad spectrum of cell lines in vitro. However, noncarcinoma cell lines possessing nonsusceptibility to NDV are rare. Here, we isolated primary mouse embryonic fibroblasts (MEFs), which are nonsusceptible to NDV. MEF-derived cells were generated by passaging the cells over fifty times to achieve spontaneous immortalization. Two of the resulting cell lines were named SLM-21 and MEF50. Karyotype analysis revealed that SLM-21 has a near-tetraploid karyotype and that MEF50 shows a near-tetraploid and near-hexaploid chimeric karyotype. NDV exerted a significant cytopathic effect on MEF50, and substantial viral replication was observed. In contrast, NDV did not have a significant effect on SLM-21, indicating that SLM-21 was a nonsusceptible cell line to NDV, while MEF50 was a susceptible cell line. The NDV authentic sialic acid (SA) receptors SA 2,3-Gal and SA 2,6-Gal were expressed in SLM-21. Transcriptomic analysis revealed that the non-susceptibility of SLM-21 may be related to its broadly activated antiviral pathways and fine-tuned regulation of the cell cycle and DNA damage. This study provides a basic cell platform for exploring viral susceptibility and pathogenesis as well as host–virus interactions during NDV infection.

TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.