Prior to a few years ago, senescent cells were a research backwater, and this state of affairs persisted for far too long given the evidence for their importance in degenerative aging. As a result, the standard assays for the presence of cellular senescence are going on twenty years old, an eternity in biotechnology development. The current thinking on senescent cells in the now revitalized field is that these methods are too crude, and that there are likely many varieties of senescence with significant differences from one another. While it is perfectly possible to build viable senolytic therapies today, producing benefits to health and longevity by selectively destroying at least some of the burden of senescent cells in old individuals, better and more comprehensive second generation therapies will require a correspondingly improved understanding of cellular senescence as a phenomenon – and certainly better assays for quantifying the presence of these cells.
Senescent cells accumulate with age, and can cause or contribute to several degenerative diseases of aging. These effects might stem from the fact that senescent cells cannot divide and therefore cannot create new cells to maintain tissue homeostasis. However, as senescent cells generally comprise a minority of cells within even very old tissues, it is more likely that senescent cells drive age-related disease cells via signaling effects. Indeed, senescent cells secrete a myriad of inflammatory cytokines, chemokines, proteases, and growth factors, the senescence-associated secretory phenotype (SASP), that can have potent effects on tissue microenvironments and thus drive age-related pathologies by mechanisms that extend beyond the loss of proliferative potential.
Traditional gene expression analyses that compare transcriptional profiles of cell populations are limited because they measure average of gene expression levels across the entire population. For example, two populations of 5000 cells each might show a twofold difference in the mRNA level of a particular gene, but this change could result from every cell expressing twice as much mRNA, or from a single cell expressing 5000 times more of that mRNA. The difference between these possibilities could have enormous phenotypic consequences in the context of a tissue. Single-cell approaches offer advantages over population studies because they can distinguish between these types of scenarios. Single-cell analyses also require fewer cells and therefore can be used to interrogate the phenotypes of rare cells, such as senescent cells produced during organismal aging.
To assess the contributions of individual senescent cells to known senescent phenotypes, we conducted quantitative PCR analyses of single quiescent and senescent cells from cultured populations of human fibroblasts. From these analyses, we find that (i) virtually all senescent cells display a gene expression signature that distinguishes them from their quiescent counterparts; (ii) nonetheless, the expression of most genes is more variable in senescent cells compared to quiescent cells; and (iii) there are correlations among genes expressed by senescent cells, including those encoding SASP factors, that localize in genomic clusters. Together, the data demonstrate that senescent phenotypes are more variable than the transcriptional profiles of cell populations previously suggested.
Identifying senescent cells at the single-cell level is an important technological step for future studies, especially in human tissues. While transgenic mouse models now allow senescent cells to be identified and isolated from mouse tissues, identifying senescent cells in human tissues remains difficult. Our findings emphasize the risk of using a single biomarker to identify senescent cells, whether in culture or in vivo. We recommend using several markers – in our own studies, for example, we tend to use combinations of SA-Bgal activity, loss of LMNB1 expression, HMGB1 relocalization, p16INK4a and/or p21WAF1 expressions, and the expression of strongly upregulated SASP factors. As many inducers of senescence (e.g., telomere attrition, ionizing radiation, bleomycin, and oncogene activation) ultimately induce a DNA damage response, it is likely that many of the factors identified in this study are common to several senescence inducers.