Cellular senescence is a state in which cells are unable to divide, either due to telomere attrition or external stress stimuli such as DNA damage or abnormal oncogene activation. Senescent cells are metabolically active and secrete inflammatory cytokines through the senescence-associated secretory phenotype (SASP). The long-term accumulation of senescent cells with age contributes to age-related conditions such as stem cell exhaustion, immunosenescence, inflammaging, and chronic diseases. Accurate identification of senescent cells in a tissue section or cell culture requires joint use of several biomarkers. Decreases in cell proliferation can be assessed by mRNA and protein levels of cell cycle regulators (p16INK4a, p21CIP1, and p53), as well as DNA damage response and repair quantified by foci analysis of double-strand break markers. Stem cell aging is driven generally by similar molecular parameters as differentiated cells. Stem cell numbers within organ tissues are difficult to count, but some research has been done on particular groups such as circulating hematopoietic stem cells. Mutations naturally occurring in blood cells during a lifetime and revealed by whole-genome sequencing can be used as a barcode for tracking and quantifying adult stem cells and their offspring in the bloodstream.Only two hematopoietic stem cell clones[56] were found, suggesting that the diversity of active stem cells is significantly depleted at an advanced age. These findings have been replicated in individuals of many ages, revealing the general trend that human hematopoiesis shifts from a highly polyclonal into a quasi-monoclonal state with increasing age.[57] The biomarkers mentioned above are widely validated but are found within the cell and therefore require cell isolation (and usually destruction) for analysis. For clinical implementation, it is more useful to rely on molecules that are secreted into the adjacent extracellular area and can be detected without disturbing the cells in human subjects. The various SASP components, such as inflammatory molecules (cytokines and chemokines, extracellular damage-associated alarmin high-mobility group box 1 [HMGB1] protein), growth factors and regulators (transforming growth factor beta [TGF], insulin-like growth factor-binding protein), formin (TAME) Biomarkers Workgroup established a framework for the selection of blood-based biomarkers for next-generation geroscience clinical trials.[65] Data on the quantity of active stem cells in other organs and how their abundance and functionality change with age would be very pertinent, especially in the context of novel therapeutic approaches, such as direct reprogramming of differentiated tissue cells[58] or rejuvenation of dysfunctional adult stem cells,[59] designed to maintain robust stem cell pools.

3.2. Inflammatory Markers

Aging is often associated with the dysregulation of the immune system, leading to chronic systemic low-grade inflammation. Circulating pro-inflammatory mediators, such as cytokines and their soluble receptors (IL-6, IL-8, IL-1, TNF-, TNFRs), C-X-C motif chemokines (CXCL9, CXCL10), C-C motif chemokines (CCL2, also known as MCP-1, CL11 also known as eotaxin), CX3C motif chemokine (CX3CL1) as well as acute phase C-reactive protein produced in liver in response to inflammation are among promising biomarkers of aging.[1] Due to the fact that many of these mediators are classical SASP components, cellular senescence is likely to participate in inflammaging, albeit the true extent to which senescence contributes to systemic markers compared to other inflammatory drivers remains unknown. Additional information regarding age-related changes in immune function can be obtained from immune-phenotype profiling (lymphocyte subset panel, CD4:CD8 T cell ratio).[6]

2.6. Altered Intercellular Communication

Although cell aging is assumed to be the main cause of overall human aging, our body consists not only of cells but also of the extracellular space, which is likewise affected by time. Cross-linking of extracellular matrix molecules, especially collagen, and accumulation of waste products, such as AGEs, make the cell environment rigid, impeding cell-cell interaction and cell migration within tissues.[60] In contrast to the intracellular space, which has efficient intracellular mechanisms of waste recycling, the extracellular space lacks such machinery and disposal of garbage outside the cell is a serious age-related problem. Regulatory and effector molecules that are secreted into an immediate environment, such as various SASP components or found in circulating body fluids such as hormones, systemic inflammatory markers, and circulating miRNAs also contribute to altered cell communication and activity with age. Despite no standard cytokine signature existing in age-related chronic inflammation in contrast to the acute inflammatory response for which a number of secreted molecules have been validated, a deep-learning-based inflammatory aging (iAGE) clock has been recently created (Table S1, Supporting Information).[66] The clock is based on circulating immune protein data of 50 cytokines, chemokines, and growth factors, with the ability to predict multimorbidity with 0.41 mean absolute error. In centenarians, iAge was on average 40 years lower than their corresponding chronological age, with the chemokine CXCL9 and down-regulated Sirtuin-3 as the strongest contributors.

3. Systemic Biomarkers