An Approach to In Vivo Detection of Senescent Cells

The accumulation of senescent cells is an important cause of aging. These cells are created in large numbers, day in and day out, but near all are quickly destroyed, either by their own programmed cell death processes, or by the immune system. A tiny fraction linger, however, and produce a potent mix of inflammatory signals that disrupt tissue function in many ways. The more senescent cells, the worse the consequences. There are assays to detect senescent cells in tissue samples, but these tests are all quite old and cumbersome, with a limited range of application. The research community would benefit greatly from improved methods of detection of senescent cells, assays capable of greater discrimination, and particularly those that can run in a living animal. The paper noted here is a step in that direction.

Although senescent cells are well-characterized in culture, identifying senescent cells in vivo has been challenging. The inability to reliably identify senescent cells in an intact organism has impaired the study of their precise role in tumor suppression and physiological aging. To date, activation of p16INK4a expression has proven to be one of the most useful in vivo markers of senescence. The expression of p16INK4a is highly dynamic, being largely undetectable in healthy young tissues, but rising sharply in many tissues with aging or after certain sorts of tissue injury. Murine studies suggest that accumulation of p16INK4a leads to an age-related loss of replicative capacity in select tissues, thereby causing some phenotypic aspects of aging.

Our laboratory and others have placed reporter genes under the control of the p16INK4a promoter by either transgenic or knock in approaches. These reporter alleles have been employed to demonstrate that the p16INK4a promoter activity increases during wounding, inflammation, tumorigenesis, or aging in vivo in tissues. While valuable for studies at the tissue or organ level, these alleles have been limited in their ability to detect and isolate individual cells with strong activation of the p16INK4a promoter in vivo. To study individual p16INK4a-activated cells, we have generated a fluorescence-based reporter allele with tandem-dimer Tomato (tdTom) knocked into the endogenous p16INK4a locus. This allele enables the identification and isolation of p16INK4a-activated cells at the single-cell level from cultured cells and in vivo. Using this allele, we quantified tdTom+ cells in several tissues with aging or in the setting of inflammation, and isolated these cells for characterization in terms of function and gene expression.

Link: https://doi.org/10.1073/pnas.1818313116

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