Cellular senescence, or the permanent loss of the ability to proliferate, occurs in somatic cells both within organisms and in cell culture. Cellular senescence is a permanent state of cell cycle arrest characterized by flat cell morphology, altered gene expression and lack of proliferation [4], and other alterations in, for example, gap junctions [5]. Senescence plays both a positive and negative biological role in organismal health. Senescence is a natural suppressor of cancer initiation by causing cell cycle arrest in potentially harmful cells. Wound healing studies have found that senescent cells are necessary for optimal wound healing [6]. Senescent cells in development secrete macrophage recruiting chemokines that lead to the removal of the senescent cells and keep the tissue functioning properly [7]. However, senescent cells can also contribute to aging and disease. The secreted factors of senescence can lead to inflammation including inflammageing [8, 9], and cause tissue dysfunction [10]. Senescent cells play a role in the progression of several diseases, including cystic fibrosis, atherosclerosis, and neurodegeneration [11-13]. In mouse models of senescence, the removal of senescent cells can rescue neurocognitive dysfunction and improve glucose metabolism [14, 15]. Understanding cellular senescence is not only critical to our understanding of basic biological processes of development, wound healing, and aging, but also provides targets for interventions for the prevention or treatment of disease [16]. Senolytics have progressed to early-phase clinical trials [17]. More recently, immunosenescence is thought to drive the ageing of solid organs [16].

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Figure 1. Cellular senescence inducers and the SASP. Molecular and physiological triggers that may lead to cellular senescence including replicative and oxidative stress lead to DNA damage, chromatin remodeling, and cell cycle arrest. The senescence-associated secretory phenotype includes multiple factors that signal via autocrine and paracrine mechanisms. [1].

The two major categories of cellular senescence are replicative senescence (RE) and stress-induced premature senescence (SIPS). Replicative senescence, first described by Hayflick and Moorhead, refers to the diminished proliferation after multiple cell divisions in culture that leads to cell cycle arrest and cellular senescence in vitro [35]. Replicative senescence is caused by telomere shortening and the associated signaling alterations in somatic cells, including ataxia telangiectasia mutated (ATM) kinase activation, which initiates cell cycle arrest [36]. This type of senescence is what leads to the aging phenotype where tissues and cells degrade over time, and ultimately result in the death of an organism. Cells in replicative senescence are different from quiescent or non-proliferating differentiated cells. Senescence includes cellular stress responses that ultimately cause irreversible arrest, resistance to apoptosis and interfere with differentiation [37]. Stress induced premature senescence (SIPS) may be caused by a plethora of stressors, including oncogene activation, DNA damage [16], loss of tumor suppressor gene function and even cell culture stress. Oncogene-induced senescence is caused by oncogene expression (OIS), while loss of tumor suppressor gene induced sentences (TSIS) is caused when a tumor suppressor gene fails to perform its typical function. Figure 1 summarizes the various triggers of the cellular senescence pathway (Fig. 1) [1].

Several cellular components and pathways contribute to senescence. Telomeres are areas on the ends of chromosomes with DNA repeats with which proteins associate. Each cell division causes a small shortening of the telomere length because DNA polymerases are unidirectional and thus unable to prime a new DNA strand. As the telomeres erode over time, they signal a DNA damage response (DDR), which initiates and maintains senescence [38]. Telomere shortening that occurs with age can be reversed by expression the enzyme telomerase, which elongates telomeric sequence and keeps the induction of senescence at bay [39]. However, most cells do not express telomerase. Other mechanisms that trigger the DDR also lead to senescence. These include DNA double strand breaks, histone deacetylase inhibitors and mitogenic signals that create DNA damage. Aneuploidy, or aberrant chromosomal number, occurs due to dysfunction of mitotic machinery in aged cells and is associated with an early senescence phenotype [40]. Mitochondria are also linked to senescence [41]. Mitochondrial dysfunction leads to insufficient energy for the cell to maintain appropriate replication [42]. Furthermore, the product of mitochondrial dysfunction, Reactive Oxygen Species (ROS), are known to elicit cellular senescence via telomere shortening [43, 44].

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Figure 2. Signaling in Senescence. Cellular senescence is induced and maintained via several signaling pathways and may of the key players including p53, p21 and p16 are also useful as biomarkers [2].

The phenotypic characteristics of senescent cells can be quite variable but common features include increased cell size, nuclei enlargement, prominent Golgi and vacuolated cytoplasm, senescence-associated β-galactosidase, altered p53 activity, and increased methylation [45-47]. Senescence leads to a characteristic cellular phenotype that includes a secretory profile. The senescence-associated secretory phenotype (SASP) is another way to identify senescent cells [48].

Senescent cells do not have markers that are entirely specific, and not all senescent cells express all possible senescence biomarkers. In general, though, the senescent state includes several features and biomarkers. First, the growth arrest of senescent cells is permanent and cannot be reversed by stimuli. Senescent cells can be twofold larger than their counterparts not in senescence, frequently have increased lysosomal size, and often have increased expression of senescence-associated β-galactosidase (SA-βGal) [49]. The most reliable detection of senescence cells utilizes multiple biomarkers simultaneously. Biomarkers may be measured my many methods including Western blot analysis, immunohistochemistry, immunofluorescence, and ELISA. Innovative methods described recently have measured biomarkers with flow cytometry, nanoparticle-based delivery of imaging agents, and positron emission tomography [50]. Finally, biomarkers typically play major rolls in the signaling events that induce, promote, and maintain senescence. Figure 2 diagrams the major signaling pathways involved in cellular senescence, many components of which will be examined below [2].

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Figure 3. Detection of DEC1 and SA-β-Gal activity in fresh tissue sections. DEC1 expression and SA-β-Gal activity in ESCC and adjacent normal epithelia were detected by immunohistochemistry and SA-β-Gal staining assay in consecutive frozen sections [3].

Possibly the most frequently used biomarker of senescence is the increased activity and expression of lysosomal β -galactosidase. Senescence-associated β-gal activity is histochemically detectable at pH 6.0 [45]. In contrast, β-gal activity in replicating cells is detectable at pH 4. Activity of the enzyme is measured by utilizing the substrate galactopyranoside with either chromogenic or fluorescent tags for detection [16, 51, 52]. Lysosomal mass is also indicative of increased β-gal levels in senescence [53]. See Figure 3 for an example of β-gal detection utilizing a staining assay in frozen tissue sections [3].

One of the major pathways involved in senesce is the p53-p21 pathway. The transcription factor p53 is involved in the regulation of over 500 target genes and modulates a range of functions including DNA repair, the cell cycle, apoptosis, and senescence. Senescent cells have increased p53 expression and activity. Overexpression and increased activity of p53 induces premature aging in mice [54]. When mitosis is delayed p53 can cause cell death or senescence. Telomere shortening can be recognized as double strand breaks in the DNA and lead to the DNA damage response (DDR), a signaling cascade involving ataxia telangiectasia-mutated (ATM) kinase. ATM activates p53 to cause cell-cycle arrest and senescence [55]. Telomere shortening is linked to increased p53 expression and activity [56].

The checkpoint protein p21 (CDKN1A) is a cyclin dependent kinase (CDK) inhibitor that is activated by p53. Activation of p21 causes the prevention of phosphorylation of the retinoblastoma protein (Rb), a tumor suppressor protein that inhibits cell cycle progression if a cell is under stress. Senescent cells have increased p21 expression. Long term activation of p21 can induce dysfunction of the mitochondria and create reactive oxygen species (ROS), which play a large role in DNA damage and the DDR and elicit or maintain the senescent phenotype [57].

The p53-binding protein 1, or 53BP1, is a tumor suppressor and a protein binding partner to p53 and acts to recruit several other proteins involved in the DNA damage response (DDR) [58]. 53BP1 recognizes unique methylation on histones and facilitates DNA damage repair, not enzymatically, but by offering a binding site and scaffold for other DDR proteins [59]. A unique characteristic of 53BP1 is that it plays a large role determining the repair pathway initiated in the DDR [60]. Functional 53BP1 is critical to several cellular mechanisms. It is required for VDJ recombination in the adaptive immune system [61]. Defective 53B1 impairs DNA repair functions and causes increased sensitivity to ionizing radiation [62]. 53BP1 mediates p53 activity to cause p-21 dependent cell cycle arrest.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1) and DEC2 are transcription factors that have roles in circadian rhythm, hypoxia response, and cell proliferation. DEC1 is a target of p53 and mediates p53-dependent senescence [63]. Stressors like hypoxia and cytokines affect DEC1/DEC2 expression which regulate tumor progression in many types of cancer cells [64-66]. Overexpression of DEC1 induces cellular senescence and inhibits cell growth in cell culture [3]. In fresh tissue sections, strongly positive expression of DEC1, as measured by immunohistochemistry, correlated with an increase SA-β-Gal activity and in the number of senescent cells (Fig 3) [3]. In addition, DEC1 expression was significantly correlated with survival of esophageal squamous cell carcinoma patients, suggesting that the increased expression of DEC1 protects the patient by the induction of cellular senescence [3]. In prostate cancer cells, induction of cellular senescence can be caused by thyroid hormone through DEC1 signaling [67]. See Figure 3 for an example of DEC1 immunohistochemical detection in frozen tissue sections [3].

The second major molecular signaling pathway linked to cellular senescence is the p16INK4a-retinoblastoma (RB) pathway. The tumor suppressor gene p16 INK4a (p16 hereafter) is a canonical cell-cycle inhibitor that inactivates cyclin-dependent kinases including CDK4 and CDK6, inducing cell cycle arrest and senescence [68, 69]. The gene CDKN21 encodes the p16 protein [70]. The expression of p16 is controlled by epigenetic regulation and transcription factors including YY1 and Id1 [71, 72]. The p16 protein is expressed more in mice and humans with age [73, 74]. The enhanced expression of p16 drives senescence in multiple cell types [75, 76]. Age-related pathologies are associated with increased p16 expression and activity. For example, patients with elevated p16 in senescent astrocytes are at increased risk for sporadic Alzheimer’s disease [77]. Signaling via p16-RB can cause senescence associated heterochromatic foci (SAHF), which silences pro-proliferative genes [78]. Interestingly, overexpression of p16 in cell culture causes cell cycle arrest and morphological features of senescence that remain even when p16 levels return to baseline [79]. Recently, flow cytometry of p16 positive cells via immunofluorescence has identified mesenchymal stem cells at risk of replicative senescence [80].

The peptide hormone GDF15, also known as macrophage inhibitory cytokine-1 (MIC-1) or non-steroidal anti-inflammatory drug activated gene-1 (NAG-1) is a cytokine that plays a role in energy metabolism and weight. The full-length protein is found in the cytoplasm as well as the nucleus, where it controls regulation of the Smad pathway [81]. However, the cleaved GDF15 peptide is secreted and found in the extracellular space and circulation in response to cellular stress or injury. Since it is secreted, GDF15 is measurable in the circulation and thus is an excellent biomarker candidate. Elevated circulating levels of GDF15 are found in many diseases including cardiovascular disease, cancer, and diabetes [82-84]. Overexpression of GDF15 in mice both reduced their body weight and had anti-tumorigenic activity [85]. The molecular mechanisms of GDF15 are not fully elucidated but its receptor was recently identified and will likely lead to the better understanding of it [86]. Knockout of GDF15 in human dermal fibroblasts caused mitochondrial dysfunction and early senescence [87]. Selective GDF15 depletion in fibroblasts in a 3D skin model led to epidermal thinning, a specific indicator of skin aging, indicating that GDF15 is involved in mitochondrial homeostasis and suppresses cellular senescence.

The secreted glycoprotein STC1 is a secreted protein involved in calcium/phosphate homeostasis and is associated with several human cancers. Decreased expression of STC1 in cancer cells promotes cell growth while overexpression of STC1 inhibits cell proliferation [88]. STC1 is an SASP factor which is measurable in plasma and STC1 correlates with age in humans [31]. Inhibition of STC1 in mesenchymal stromal cells decreases proliferation and causes elevated ROS generation [89].

SerpinB2, also known as plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that has elevated expression in senescent cells [90]. SerpinB2 plays a role in senescence by stabilizing p21 in senescent cells [32]. SerpinB2 can also lead to senescence when overexpressed in human fibroblasts [32]. SerpinB2 is a mediator of the fibrolytic system and may contribute to the modulation of platelet activity by senescent cells [33].

The lack of proliferation by senescent cells is correlated with reduced rate of DNA synthesis as compared to that of proliferating cells. In fact, DNA synthesis rates are known to decrease in senescence and this decrease is correlated with an increase of SA-β-gal activity [91]. In addition, both pharmacological and genetic inhibition of DNA nucleotide synthesis can induce premature senescence in human mammary epithelial cells [92]. Detection of DNA synthesis may be accomplished by utilizing 5-ethynyl-2’-deoxyuridine (EdU) that is labeled with a fluorescent azide for DNA incorporation. This assay provides sensitive and quick results in tissue and organ samples. This technique is quicker and easier than Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU) incorporation assays to measure DNA synthesis rates because it does not require the fixation of samples or DNA denaturation [20]. Combining the use of biomarker expression levels with cell proliferation rates provides more reliable evidence of senescent cells than utilizing either individually.

A common characteristic of DNA damage induced senescent cells is that they have altered secretion profiles, referred to as the Senescence-Associated Secretory Phenotype (SASP). Transient SASP contributes to tissue homeostasis but chronic SASP is a key driver of chronic inflammation and many of the pathological symptoms of aging and even cancer [93, 94]. Interestingly, the SASP of cells with different stimuli that led to senescence is different. Stress-induced premature senescence and replicative senescence share a common SASP profile but cells induced into senescence by proteasome inhibition secrete different compounds [95]. Recently, proteomic approaches have led to the recognition of components of the SASP profile in relation to the way senescence was induced, especially those that overlap with markers of aging in human plasma [31]. The SASP includes both soluble and insoluble components including soluble signaling factors, secreted proteases, and secreted but insoluble proteins [96]. Recently, the rasGAP SH33-binding protein 1 (G3BP1) was shown to be required for the SASP [97]. Interestingly, depletion or inhibition of G3BP1 prevents the expression of SASP components but does not stop senescence and these SASPless senescent cells do not promote cancer cell growth like typical senescent cells do [97].

The effects of SASP factors on the regenerative capacity of tissues were studied using intestinal organoids as a model [98]. The authors found that SASP factors secreted by senescent fibroblasts impair stem cell functions and crypt formation. The N-terminal domain of Ptk7 was demonstrated to be the main component of the SASP that upregulates non-canonical Wnt / Ca2+ signaling through FZD7 in stem cells. Changes in cytosolic [99] induced by Ptk7 activate nuclear translocation of YAP and expression of YAP/TEAD target genes, suppressing stem cell differentiation.

A recent study found that both apoptosis and senescence are regulated via mitochondria-related mechanisms, and mitochondrial apoptotic stress is the main activator of the SASP [100]. Mitochondrial outer membrane permeabilization (MOMP) was detected in a subset of mitochondria as a marker of cellular senescence. This mechanism, which is named minority MOMP, involves BAX and BAK macropores and activates the translocation of mitochondrial DNA (mtDNA) into the cytosol, followed by the stimulation of the cGAS-STING pathway, a major activator of the SASP. Furthermore, suppression of MOMP decreased inflammation in aged mice.

Interleukin-6 (IL-6) is the most prominent cytokine of the SASP. This cytokine is proinflammatory and is associated with DNA damage-induced senescence [101]. The secretion of IL-6 in senescent cells is regulated by persistent DNA-damage signaling through ATM and CHK2 [102]. IL-6 is a proinflammatory cytokine with multiple functions. The role IL-6 plays in senescence can be paracrine to support tumorigenesis and cell proliferation or by triggering an immune response and exerting tumor suppressive functionality [103].

Interluekin-8 (IL-8) is a chemokine secreted by blood monocytes, fibroblasts, and senescent cells with SASP [104]. IL-8 was originally knowns as neutrophil activating factor (NAF) or monocyte-derived neutrophil chemotactic factor (CXCL8). Expression and secretion of IL-8 is stimulated by several stressors such as reactive oxygen species (ROS) or environmental conditions [105]. Secreted IL-8 recruits neutrophils and is a key component of the host inflammatory response [106]. In senescence, persistent DNA damage causes IL-8 secretion [102].

The protein p27KIP1 is a cyclin-dependent kinase inhibitor whose expression is increased in senescent cells. Ectopic expression of p27 in mouse embryo fibroblasts causes permanent arrest of the cell cycle and a senescent-like phenotype [107]. Situin6 expression decreases p27 levels by inducing its degradation and thereby antagonizes senescence [108]. Induction of senescence via RB causes an accumulation of p27 and the ability of RB to induce sentence is reversible by eliminating p27 expression [109]. A recent study demonstrated that depletion of RNA Binding Motif Protein 4 (RBM4) induced P27-dependent senescence and proliferation arrest via LKB1-AMPK-mTOR cascade [110]. In particular, RBM4 interfered with the LKB1/STRAD/MO25 heterotrimeric complex by binding LKB1 and induced LKB1 ubiquitination and degradation in nucleus. The study showed that RBM4 is involved in bypassing senescence and sustaining cell proliferation.

Methylation is a common reversible modification to histones which bind to DNA [111, 112]. Methylation may result in silencing of genes or increased transcriptional activation [113]. In cultured cells senescence is associated with genome-wide DNA hypomethylation and focal hypermethylation [18]. Chronic oxidative stress causes epigenetic changes that lead to cellular senescence. Premature senescence induced by oxidative stress as well as replicative senescence have global hypomethylation although they have differences in the methylation enzyme expression [114].

The length of telomeres is a predicator of the ability of a cell to replicate [115]. The onset of replicative senescence is triggered by telomeres reaching a critically short length that causes a DNA damage response with p53 activation and upregulation of p21 [116]. Telomere shortening is correlated with the accumulation of senescence-associated heterochromatin foci (SAHF) [78]. The highly dense heterochromatin bodies are transcriptionally inactive but have increased chromatin-bound high mobility group A2 (HMGA2) [117]. SAHF include a histone H2A variant (macroH2A) and heterochromatin protein 1 (HP1) proteins [21, 118]. The histone chaperone Ant-silencing function 1a (ASF1a) functions in chromatin assembly and transcriptional regulation. ASF1a is necessary for telomerase expression and reduced expression of ASF1a leads to growth arrest and senesces via an increase in DNA damage and upregulation of p53-p21 signaling [119]. Histone γ-H2AX is a replacement histone that is phosphorylated at C-terminal serine-139 and whose strong association with DNA double strand breaks gives it great utility as a biomarker of senescence [120]. In senescent cells histone γ-H2AX foci are found to correlate with telomere dysfunction-induced foci [121]. However, histone γ-H2AX foci also associate with non-senescent cellular DNA double strand breaks, especially those from ionizing radiation [122]. DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS) are found in senescent cells that have persistent DDR signaling and nuclear foci. The foci include activated DDR proteins such as phosphor-ATM and the stabilizing component histone H2AX [123]. DNA-SCARS differ from reversible DNA damage foci in that the DNA damage is irreversible.

DNA damage and the accumulation of the DNA damage machinery, specifically 53BP1, at uncapped telomeres is referred to as telomere dysfunction induced foci or TIF [124]. Telomere uncapping happens in cells with critically short telomeres or when protective telomere factors are not functioning. Uncapped, dysfunctional telomeres may be caused by inhibition of telomeric repeat-binding factor 2 (TRF2), which is typically a telomere protector [125]. Uncapped telomeres are associated with DNA damage response factors like 53BP1 and ɣ-H2AX and this provides a good method for TIF detection with antibodies to either protein [124, 126]. The detection of TIFs indicate telomere dysfunction, a hallmark step in cellular senescence.

The reduction of the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice induced cellular senescence, inflammatory signaling and oxidative stress, causing endothelial dysfunction. Moreover, telomere dysfunction-activated senescence decreased glucose tolerance via induced inflammation in the liver and adipocytes and suppressed microvascular density [127].

Chronic nuclear DNA damage leads to a DNA damage response (DDR) that functions to arrest the cell-cycle to allow a healthy cell to remove or repair the damage. Long term DDR is associated with cellular senescence and is known to activate proteins including p53 [128]. DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS) are found in senescent cells that have persistent DDR signaling and nuclear foci [123]. The foci include activated DDR proteins such as phosphor-ATM.

As the area of cellular senescence research has expanded over the years more and more methods for studying the mechanisms involved have been developed.

One of the most important methods used to study cellular senescence is cell culture of mammalian cell types. Culturing cells in vitro is an excellent model that is easily manipulated and examined at the molecular level. Stressors such as radiation, genotoxic drugs, and oxidative stressors can be introduced into cells to induce senescence or cells may be grown long-term in culture to lead to replicative senescence [129, 130]. Treatment of non-transformed cells with ionizing radiation causes DNA damage, G2 cell cycle arrest, and ultimately cellular senescence [131]. Non-ionizing radiation such as ultraviolet irradiation also causes oxidative damage that may lead to senescence in fibroblasts [132]. Genotoxic drugs cause extensive DNA damage but many common cancer treatments are genotoxic. Cisplatin and doxorubicin are two commonly used cancer treatments that can induce senescence in tumor cells [133, 134]. Oxidative stress also leads to cellular senescence and can be caused by radiation and reactive oxygen species.

A vast array of mouse models has been developed that help study cellular senescence. The senescence accelerated mouse, called SAMP8, is a spontaneous model of oxidative damage and amyloid precursor protein (APP) overproduction [135]. These mice exhibit oxidative stress and neurological symptoms such as memory loss. SAMP8 mice have proven to be an excellent model for testing therapeutics for aging related cognitive decline [136, 137]. In a progeroid mouse model the selective killing of p16-positive senescent cells reduces aging-related features [138]. A mouse model lacking telomerase has phenotypic markers of aging like osteoporosis and hair graying [139].

Telomeres are DNA sequences at the ends of eukaryotic chromosomes that contain TTAGGG repeats and bind to shelterin proteins and RNA that help protect them from DNA damage detection in the cell. Telomeric DNA diminishes in dividing cells over time since DNA polymerase continue duplication to the end of the chromosome, known as incomplete semi-conservative DNA replication. Some DNA bases of the telomere are truncated with each DNA duplication event. Over a person’s lifetime, telomeric DNA sequence may decrease by more than half. However, this protects other genes within the chromosome from being truncated or deleted in the replication process. Telomerase is an enzyme that can replenish telomeric DNA in eukaryotic cells under certain states, such as in development, cancer or in dividing stem cells.

Several techniques are available to study both the telomeric DNA and the telomerase protein. Telomere Restriction Fragment (TRF) Analysis is used to determine the average telomere length in cells [24]. This technique modifies the Southern blot to determine the lengths of terminal restriction fragments. This relies on the lack of restriction enzyme recognition sites within TTAGGG tandem repeats in the telomeric regions that keep telomeric but not genomic DNA from being digested. Telomere dysfunction Induced Foci (TIF) analysis measures DNA damage foci at uncapped telomeres by co-localization of DNA damage and shelterin proteins which associate with telomeres [126]. Telomerase Repeated Amplification Protocol (TRAP) measures telomerase activity by extension, amplification, and detection of telomerase products via electrophoresis [140].

Biomarkers of cellular senescence are measured with a plethora of techniques. Western blots and Enzyme Linked Immunosorbent Assays (ELISA) are widely utilized for measurements of biomarkers in samples from cell culture to patients. The secreted SASP related IL-6 is just one example of a biomarker measured by ELISA [141]. Southern blot or quantitative PCR are used to measure telomere length from DNA preparations [142]. Quantitative Real-Time PCR (qRT-PCR) is highly utilized for the measurement of mRNA for biomarkers from RNA preparations. New approaches to the detection of senescence biomarkers are continually being developed. One recent technique utilizes single-cell RNA sequencing for the comparison of biomarker signatures of cellular senescence with cell type based on the gene expression profile detected. This method recently showed that aged mice had a 10% increase in senescent endothelial cells in mouse cerebral microcirculation [143].

The identification of cellular senescence is critical for researching the basic biology of senescence as well as for the development of detection, diagnostic, and therapeutic techniques. Since a universal marker of cellular senescence has not yet been found, efforts are being made to discover more specific biomarkers. One interesting aspect of cellular senescence is while it is almost universal in somatic cells of animal species some invertebrates like sea urchins do not undergo cellular senescence [144]. Understanding the mechanisms behind this lack of senescence and its implications may build an area of research that leads to important understanding of the senescence process and outcomes. Recently an age dependent change in transcription in the red sea urchin demonstrated an upregulation of genes involved in neuroprotection as well as autophagy and proteasome function, which may be important to mitigating the effects of aging [145].

Recently, senescent cells have been shown to possess a heat-shock and autophagy-dependent ability to resist stress granule formation [146]. Stress granules are related to neurodegenerative disease associated pathological granules. Understanding the ability of senescent cells to prevent stress granule formation has the potential to further the understanding of human diseases caused by similar granules such as Parkinson’s and Alzheimer’s diseases. Senescence is also being studied for its potential role in Duchenne muscular dystrophy [147]. Together, these recent studies demonstrate that more and more aging related human diseases are intimately linked with cellular senescence and further studies will eventually elucidate and hopefully utilize the mechanisms involved for the betterment of human health and longevity.

Cellular senescence may be involved in the increased potential of COVID-19 to cause morbidity and mortality in the elderly [148]. In patients with COVID-19, gene expression changes promoting inflammation and cellular senescence occur in the peripheral blood mononuclear cells [149]. Leukocytes with short telomere lengths are associated with the more severe cases of COVID-19, and the immune response necessary to overcome the disease requires expansion the lymphocyte population which is a process depending on telomere length [150].

Cellular senescence is intimately linked with inflammation, aging, and several other diseases. Neurodegenerative diseases such as Alzheimer’s disease have tau accumulation and neurofibrillary tangles. Tau has been shown to have a positive relationship with cellular senescence and may be a worthwhile target for intervention therapies [14, 151]. In senile osteoporosis, which affected aged males and females, bone marrow stromal cells differentiate into adipocytes and undergo senescence, leading to decreased bone formation and frail bones [152]. Interventions to decrease the number of bone marrow stromal cells that undergo senescence may lead to clinically helpful therapies. Joint health also diminishes with age and serine proteinase targeted therapy may help with cartilage in the joint [153]. Endothelial cellular senescence causes cardiovascular disease and atherosclerosis but is inhibited by nitric oxide [154]. Increasing the bioavailability of nitric oxide or the activity of endothelial nitric oxide synthase positively affect telomerase activity [155]. The SASP is also an attractive target for the development of therapies, as many of the secreted proteins modulate the microenvironmental conditions senescent cells exist within [156]. In summation, many key mediators of senescence are promising targets for therapies to treat aging related disease.

Several human cancers have upregulation of the histone chaperone ASF1a, which provides them infinite proliferation potential [119]. Since diminished ASF1a levels are associated with senescence, therapies to decrease ASF1a in cancers have potential may be a potential target for future cancer therapeutics. The SASP of senescent cells wreaks havoc on the microenvironment and is pro-oncogenic. Klotho protein may inhibit the SASP [157] which may be useful in developing therapies for aging related and inflammatory diseases. Cancer cells almost universally have telomere maintenance unlike their somatic cell counterparts, making telomere maintenance a potential target for cancer therapies. Immunotherapies, telomerase inhibitors, and promoter mutations in the telomerase gene are all being studied towards the goal of cancer treatment.

Cells that escape senescence may acquire immortalization which is a critical step in both cancer development and creating immortalized cell lines to use for in vitro research. Primary cells from non-cancerous tissues have finite lifespans and do not proliferate indefinitely in culture due to replicative senescence [35]. Several events may cause a primary cell to avoid senescence and become immortal. These include stabilization of telomere length and gene silencing by methylation. Genetic modifications or optimization of the culture environment are necessary to artificially immortalize primary cells in culture. Immortalized cells may be useful in therapies to treat the myriad of aging and senescent related diseases. Culture optimization may alter oxygen levels or cell density or may introduce growth factors. One promising approach is matrix optimization, which can utilize decellularized cell-deposited extracellular matrix (dECM) to condition stem cells from patients [2].

Cellular senescence is both an intriguing culmination of the life of a cell and a nexus of cellular events that lead an organism to aging and disease. To understand the mechanisms that cause cellular senescence is to also create a list of opportunities for therapeutics for aging and diseases associated with senescence. This area of research holds a great deal of promise for clinical improvements to the human condition. For example, the link between senescence and cancer is strong and senescence can stop premalignant cell proliferation. However, senescent cells can also lead to chronic inflammation and thereby cause a pro-tumorigenic environment. Therefore, one large area of future work and innovation is within the clinic. While many methods of detection of senescence utilize fixed or frozen tissues, diagnostic tests that are performed in real time are being developed to improve clinical diagnoses and care. Strategies to utilize flow cytometry, PET, and measurement of circulating factors may improve the detection and speed of senescent cell measurement and provide clinicians with data quickly to speed their ability to offer treatment strategies to patients [50]. Targeting senescent cells as a therapeutic strategy is likely to also blossom in the coming years [158]. Utilization of senolytic agents shows promise in inhibiting senescent cells and the associated pathologies [159]. Furthermore, the SASP of senescent cells may be a useful therapeutic target for immunotherapeutic approaches [160].