Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.     

Expression of coxsackie and adenovirus receptor distinguishes transitional cancer states in therapy-induced cellular senescence

Therapy-induced cellular senescence describes the phenomenon of cell cycle arrest that can be invoked in cancer cells in response to chemotherapy. Sustained proliferative arrest is often overcome as a contingent of senescent tumor cells can bypass this cell cycle restriction. The mechanism regulating cell cycle re-entry of senescent cancer cells remains poorly understood. This is the first report of the isolation and characterization of two distinct transitional states in chemotherapy-induced senescent cells that share indistinguishable morphological senescence phenotypes and are functionally classified by their ability to escape cell cycle arrest. It has been observed that cell surface expression of coxsackie and adenovirus receptor (CAR) is downregulated in cancer cells treated with chemotherapy. We show the novel use of surface CAR expression and adenoviral transduction to differentiate senescent states and also show in vivo evidence of CAR downregulation in colorectal cancer patients treated with neoadjuvant chemoradiation. This study suggests that CAR is a candidate biomarker for senescence response to antitumor therapy, and CAR expression can be used to distinguish transitional states in early senescence to study fundamental regulatory events in therapy-induced senescence.     

Senescent skeletal muscle fibroadipogenic progenitors recruit and promote M2 polarization of macrophages

Senescent cells compromise tissue structure and function in older organisms. We recently identified senescent fibroadipogenic progenitors (FAPs) with activated chemokine signaling pathways in the skeletal muscle of old mice, and hypothesized these cells may contribute to the age-associated accumulation of immune cells in skeletal muscle. In this study, through cell–cell communication analysis of skeletal muscle single-cell RNA-sequencing data, we identified unique interactions between senescent FAPs and macrophages, including those mediated by Ccl2 and Spp1. Using mouse primary FAPs in vitro, we verified increased expression of Ccl2 and Spp1 and secretion of their respective proteins in the context of both irradiation- and etoposide-induced senescence. Compared to non-senescent FAPs, the medium of senescent FAPs markedly increased the recruitment of macrophages in an in vitro migration assay, an effect that was mitigated by preincubation with antibodies to either CCL2 or osteopontin (encoded by Spp1). Further studies demonstrated that the secretome of senescent FAPs promotes polarization of macrophages toward an M2 subtype. These data suggest the unique secretome of senescent FAPs may compromise skeletal muscle homeostasis by recruiting and directing the behavior of macrophages.     

衰老相关新基因CSIG的cDNA克隆和功能

为了获得 2BS细胞衰老过程中表达下降的差异基因片段Y6 2的编码序列 ,以cDNA末端快速扩增法获得细胞衰老相关新基因CSIG(cellularsenescenceinhibitedgene ,细胞衰老抑制基因 )的cDNA全长 .CSIGcDNA长 196 1bp ,编码 4 90个氨基酸 ,在多种重要组织中都有不同程度的表达 ;蛋白产物位于细胞核内特定位点 ,可能在核仁中聚集 .细胞转染表明 :CSIG可抑制细胞衰老并延长细胞寿限 ,可能通过核糖体生物合成过程或基因转录调节来调控细胞衰老过程     

清除衰老细胞在衰老与老龄化相关疾病中的研究进展

衰老是一个新兴的重要研究领域,随着该领域相关知识的积累和技术的进步,人们逐渐意识到衰老本身可以被针对性地干预,实现延长寿命并且延缓衰老相关疾病的发生发展,具有重要的科学和现实意义.引起个体衰老的众多因素中,衰老细胞的积累被认为是导致器官衰老发生退行性变,最终引起衰老相关疾病的重要原因.近年来,多项研究表明,清除体内衰老细胞可以延缓多种衰老相关疾病的发生,直接证明了衰老细胞是导致衰老相关疾病的重要原因之一,为治疗衰老相关疾病提供了新靶点.细胞衰老是由于损伤积累诱发了细胞周期抑制通路的激活,细胞永久地退出细胞增殖周期.衰老细胞会发生细胞形态、转录谱、蛋白质稳态、表观遗传以及代谢等系列特征的改变,同时衰老细胞对凋亡发生抵抗从而在体内多器官组织积累.衰老细胞会激活炎症因子分泌通路,导致组织局部非感染性炎症微环境,进而导致器官退行性变及多种衰老相关疾病的发生发展.因此针对衰老细胞对凋亡抵抗的特性,多个研究小组通过筛选小分子化合物库,发现某些化合物能够选择性清除衰老细胞,这些小分子化合物被称为"senolytics",意为"衰老细胞杀伤性化合物".衰老细胞杀伤性化合物在多种衰老相关疾病动物模型中能够延缓疾病的发展并延长哺乳动物寿命.因此,靶向杀伤衰老细胞对多种衰老相关疾病的治疗从而提高健康寿命具有重要的临床应用前景.除靶向杀伤衰老细胞策略以外,干细胞移植、基因编辑、异体共生等策略在抗衰老研究发展中也具有重要意义,具有启发性.本文通过汇总近期衰老细胞清除领域的重要进展和多种抗衰老策略,将细胞衰老研究发展史做简要梳理,就细胞衰老与衰老相关疾病的关系作一综述,重点讨论衰老细胞在多种衰老相关疾病中作为治疗靶点的应用潜力,并就其局限性和进一步的研究方向进行探讨.     

Stress granules counteract senescence by sequestration of PAI‐1

Cellular senescence is a physiological response by which an organism halts the proliferation of potentially harmful and damaged cells. However, the accumulation of senescent cells over time can become deleterious leading to diseases and physiological decline. Our data reveal a novel interplay between senescence and the stress response that affects both the progression of senescence and the behavior of senescent cells. We show that constitutive exposure to stress induces the formation of stress granules (SGs) in proliferative and presenescent cells, but not in fully senescent cells. Stress granule assembly alone is sufficient to decrease the number of senescent cells without affecting the expression of bona fide senescence markers. SG‐mediated inhibition of senescence is associated with the recruitment of the plasminogen activator inhibitor‐1 (PAI‐1), a known promoter of senescence, to these entities. PAI‐1 localization to SGs increases the translocation of cyclin D1 to the nucleus, promotes RB phosphorylation, and maintains a proliferative, non‐senescent state. Together, our data indicate that SGs may be targets of intervention to modulate senescence in order to impair or prevent its deleterious effects.     

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.     

Clearance of senescent cells reverts the cigarette smoke-induced lung senescence and airspace enlargement in p16-3MR mice

Cigarette smoke (CS) leads to increased oxidative stress, inflammation, and exaggerated senescence, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). While the role of cellular senescence in COPD is known, it is not clear if the removal of senescent cells could alleviate the disease symptoms. To test this, we used the novel mouse model-p16-3MR, and studied the effect of ganciclovir (GCV)-mediated removal of senescent cells after chronic CS (3 months) and environmental tobacco smoke (ETS) (6 months) exposure to CS. Our results showed the reversal of CS-induced cellular senescence on the clearance of p16⁺ senesced cells by GCV treatment. Interestingly, the clearance of p16⁺ senescent cells via GCV led to a decrease in the neutrophil counts in the BALF of GCV-treated CS-exposed p16-3MR mice, as well as reversal of CS-mediated airspace enlargement in p16-3MR mice. Mice exposed to low dose ETS caused insignificant changes in the SA-β-Gal⁺ senescent cells and airspace enlargement. Overall, our data provide evidence for the role of lung cellular senescence on smoke exposure and clearance of senescent cells in p16-3MR mice in the reversal of COPD/emphysema pathology with a possibility of senolytics as therapeutic interventions in COPD.     

Senescent cells at the crossroads of aging,disease, and tissue homeostasis

Originally identified as an outcome of continuous culture of primary cells, cellular senescence has moved beyond the culture dish and is now a bona fide driver of aging and disease in animal models, and growing links to human disease. This cellular stress response consists of a stable proliferative arrest coupled to multiple phenotypic changes. Perhaps the most important of these is the senescence-associated secretory phenotype, or senescence-associated secretory phenotype —a complex and variable collection of secreted molecules release by senescent cells with a number of potent biological activities. Senescent cells appear in multiple age-associated conditions in humans and mice, and interventions that eliminate these cells can prevent or even reverse multiple diseases in mouse models. Here, we review salient aspects of senescent cells in the context of human disease and homeostasis. Senescent cells increase in abundance during several diseases that associated with premature aging. Conversely, senescent cells have a key role in beneficial processes such as development and wound healing, and thus can help maintain tissue homeostasis. Finally, we speculate on mechanisms by which deleterious aspects of senescent cells might be targeted while retaining homeostatic aspects in order to improve age-related outcomes.     

Is exercise a senolytic medicine? A systematic review

Cellular senescence, a state of irreversible growth arrest triggered by various stressors, engages in a category of pathological processes, whereby senescent cells accumulate in mitotic tissues. Senolytics as novel medicine against aging and various diseases through the elimination of senescent cells has emerged rapidly in recent years. Exercise is a potent anti‐aging and anti‐chronic disease medicine, which has shown the capacity to lower the markers of cellular senescence over the past decade. However, whether exercise is a senolytic medicine for aging and various diseases remains unclear. Here, we have conducted a systematic review of the published literature studying the senolytic effects of exercise or physical activity on senescent cells under various states in both human and animal models. Exercise can reduce the markers of senescent cells in healthy humans, while it lowered the markers of senescent cells in obese but not healthy animals. The discrepancy between human and animal studies may be due to the relatively small volume of research and the variations in markers of senescent cells, types of cells/tissues, and health conditions. These findings suggest that exercise has senolytic properties under certain conditions, which warrant further investigations.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

Exploring the emerging bidirectional association between inflamm-aging and cellular senescence in organismal aging and disease

There is strong evidence that most individuals in the elderly population are characterized by inflamm-aging which refers to a subtle increase in the systemic pro-inflammatory environment and impaired innate immune activation. Although a variety of distinct factors are associated with the progression of inflamm-aging, emerging research is demonstrating a dynamic relationship between the processes of cellular senescence and inflamm-aging. Cellular senescence is a recognized factor governing organismal aging, and through a characteristic secretome, accumulating senescent cells can induce and augment a pro-inflammatory tissue environment that provides a rationale for immune system-independent activation of inflamm-aging and associated diseases. There is also accumulating evidence that inflamm-aging or its components can directly accelerate the development of senescent cells and ultimately senescent cell burden in tissues in a likely vicious inflammatory loop. The present review is intended to describe the emerging senescence-based molecular etiology of inflamm-aging as well as the dynamic reciprocal interactions between inflamm-aging and cellular senescence. Therapeutic interventions concurrently targeting cellular senescence and inflamm-aging are discussed and limitations as well as research opportunities have been deliberated. An effort has been made to provide a rationale for integrating inflamm-aging with cellular senescence both as an underlying cause and therapeutic target for further studies.     

On the evolution of cellular senescence

The idea that senescent cells are causally involved in aging has gained strong support from findings that the removal of such cells alleviates many age‐related diseases and extends the life span of mice. While efforts proceed to make therapeutic use of such discoveries, it is important to ask what evolutionary forces might have been behind the emergence of cellular senescence, in order better to understand the biology that we might seek to alter. Cellular senescence is often regarded as an anti‐cancer mechanism, since it limits the division potential of cells. However, many studies have shown that senescent cells often also have carcinogenic properties. This is difficult to reconcile with the simple idea of an anti‐cancer mechanism. Furthermore, other studies have shown that cellular senescence is involved in wound healing and tissue repair. Here, we bring these findings and ideas together and discuss the possibility that these functions might be the main reason for the evolution of cellular senescence. Furthermore, we discuss the idea that senescent cells might accumulate with age because the immune system had to strike a balance between false negatives (overlooking some senescent cells) and false positives (destroying healthy body cells).     

Lamin B1 loss is a senescence-associated biomarker

Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-κB, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.     

Cellular senescence in osteoarthritis pathology

Cellular senescence is a state of stable proliferation arrest of cells. The senescence pathway has many beneficial effects and is seen to be activated in damaged/stressed cells, as well as during embryonic development and wound healing. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been implicated in the pathogenesis of many age‐related diseases. Osteoarthritis (OA), a severely debilitating chronic condition characterized by progressive tissue remodeling and loss of joint function, is the most prevalent disease of the synovial joints, and increasing age is the primary OA risk factor. The profile of inflammatory and catabolic mediators present during the pathogenesis of OA is strikingly similar to the secretory profile observed in ‘classical’ senescent cells. During OA, chondrocytes (the sole cell type present within articular cartilage) exhibit increased levels of various senescence markers, such as senescence‐associated beta‐galactosidase (SAβGal) activity, telomere attrition, and accumulation of p16ink4a. This suggests the hypothesis that senescence of cells within joint tissues may play a pathological role in the causation of OA. In this review, we discuss the mechanisms by which senescent cells may predispose synovial joints to the development and/or progression of OA, as well as touching upon various epigenetic alterations associated with both OA and senescence.     

The struggle of a good friend getting old: cellular senescence in viral responses and therapy

Cellular senescence is a state of stable cell cycle arrest associated with macromolecular alterations and secretion of pro‐inflammatory cytokines and molecules. Senescence‐associated phenotypes restrict damage propagation and activate immune responses, two essential processes involved in response to viral infections. However, excessive accumulation and persistence of senescent cells can become detrimental and promote pathology and dysfunctions. Various pharmacological interventions, including antiviral therapies, lead to aberrant and premature senescence. Here, we review the molecular mechanisms by which viral infections and antiviral therapy induce senescence. We highlight the importance of these processes in attenuating viral dissemination and damage propagation, but also how prematurely induced senescent cells can promote detrimental adverse effects in humans. We describe which sequelae due to viral infections and treatment can be partly due to excessive and aberrant senescence. Finally, we propose that pharmacological strategies which eliminate senescent cells or suppress their secretory phenotype could mitigate side effects and alleviate the onset of additional morbidities. These strategies can become extremely beneficial in patients recovering from viral infections or undergoing antiviral therapy.     

Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, e.g. atherosclerosis. In this study, induction of Angiotensin II (Ang II) promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cell, and depressed cell proliferation. Apoptotic changes were increased in senescent cells, with a small subset of the senescent cells showing aberrant morphology such as pronounced nuclear fragmentation or multiple micronuclei. The results suggest cell apoptosis is possibly an important factor in the process of pathologic and physiologic senescence of endothelial cells as well as vascular aging.