At the cellular level, the accumulation of macromolecular dam- age, primarily in DNA, can induce a permanent state of cellular senescence.[36] Senescent cells are characterized by an inability to divide due to stable growth arrest, driven by telomere attrition (replicative senescence) or by external stress stimuli such as DNA damage or abnormal activation of oncogenes (stress-induced premature senescence).[23] These cells demonstrate increased metabolic activity and can exert paracrine and likely endocrine ef- fects through an altered secretion profile, termed the senescence- associated secretory phenotype (SASP), which is characterized by an enrichment in inflammatory cytokines.[37] Pro-inflammatory signaling in senescent cells is promoted by the activation of the STING-mediated cytosolic DNA sensing pathway[38] due to the presence of double-stranded DNA sequences in the cytosol, such as chromosome fragments released from nuclei or LINE-1 trans- posable element transcripts.[39] It is assumed that the long-term accumulation of senescent cells with age impacts tissue renewal capacity and contributes to age-related conditions such as stem cell exhaustion, immunosenescence, inflammaging, and various chronic diseases.[40] Cell senescence has long been viewed as a tu- mor suppressive pathway; yet, SASP factors might also cause the malignant transformation of surrounding cells and hence pro- mote tumor formation.[23,41]

Given the asynchrony and heterogeneity of cellular senes- cence, and the lack of specificity of individual biomarkers, accu- rate identification of senescent cells in a tissue section or cell cul- ture requires joint use of several biomarkers, each of which is in- dicative of distinct aspects of cellular aging. Comprehensive and systematic classification and staging of organ and tissue senes- cence, as well as pathologic atrophy, remodeling, calcification, and metabolic dysfunction, are urgently needed; this was recently proposed by a working group of prominent aging researchers.[50] Scoring systems may facilitate the development of personalized treatment recommendation, such as senolytic interventions act- ing on senescing tissues, or other therapies that take into account organ-specific disease staging differentials.

2.5. Decreased Stemness

The unique features of senescent cells represent the basis for their identification (and perhaps elimination in future) by spe- cific biomarkers. The morphological changes, which can be de- tected, include large size, flattened shape, and a disorganized nu- clear envelope (mediated by reduced expression of LaminaB1).[23] 𝛽 𝛽 Lysosomal -galactosidase ( -gal) activity is the most frequently applied molecular biomarker of senescence in culture and tissue samples,[42] which reflects accumulation of junk macromolecules and aggregates in non-dividing, but metabolically active cells; 𝛽 plasma -gal activity has been recently proposed as a potential systemic biomarker of aging.[43] Decreases in cell proliferation can be assessed by mRNA and protein levels of cell cycle reg- ulators (p16INK4a, p21CIP1, and p53), however these methods in- completely distinguish senescent cells from other types of non- dividing cells such as quiescent cells (that occur due to lack of nu- trition and growth factors) or terminally differentiated cells.[23,44] The DNA damage response and repair can be quantified by foci 𝛾 analysis of double-strand break markers -H2AX and 53BPI(p53- binding protein 1), as well as by other recruited factors.[2,45] The formation of senescence-associated heterochromatic foci (SAHF) enriched for macroH2A and H3K9Me3 histone modifications, as well as members of the heterochromatin protein 1 (HP1) family, is a promising epigenetic biomarker, which reflects the silencing of genes implicated in cell proliferation.[46]

Stem cell aging is driven generally by similar molecular param- eters as the ones discussed earlier for differentiated cells. The fate of stem cells is influenced by both circulating regulatory fac- tors and the stem cell niche, consisting of mesenchymal stem cells, differentiated tissue cells, and extracellular matrix; stem cell niche deterioration is shown to be one of the leading causes of stem cell exhaustion.[51] Depending on stimuli and tissue-type, stem cells can transition into a non-proliferative quiescent state and become again activated in response to extrinsic signals, enter into irreversible senescence state and lose proliferative capacity permanently, or die by different mechanisms (Figure 1).[52]

Counting stem cell numbers within organ tissues is fraught with obvious difficulties, but for particular groups, such as cir- culating hematopoietic stem cells, some important research has been done. The hematopoietic stem cell population size ap- pears to grow steadily in early life and reaches a stable plateau by adolescence[53]; and then tends to decline with advancing age.[54] In the oldest old (> 80 years), the number of circulating + hematopoietic stem and progenitor cells (CD34 cell count) are better predictors of lifespan and cardiovascular disease-related mortality as compared to classic cardiovascular risk factors.[55] Mutations naturally occurring in blood cells during a lifetime and revealed by whole-genome sequencing, can be used as a bar- code for tracking and quantifying adult stem cells and their off- springs in the bloodstream. This technique has been applied to demonstrate that all peripheral white blood cells of a 115-year-old woman were the offspring of only two hematopoietic stem cell clones[56] suggesting that the diversity of active stem cells is sig- nificantly depleted at this advanced age. These findings have been replicated in individuals across many ages, revealing the general trend that human hematopoiesis shifts from a highly polyclonal into a quasi-monoclonal state with increasing age.[57]