SignificanceThe accumulation of senescent cells over a lifetime causes age-related pathologies; however, the inability to reliably identify senescent cells in vivo has hindered clinical efforts to employ this knowledge as a means to ameliorate or reverse aging. Here, we describe a reporter allele, p16tdTom, enabling the in vivo identification and isolation of cells featuring high-level activation of the p16INK4a promoter. Our findings provide an insight into the functional and molecular characteristics of p16INK4a-activated cells in vitro and in vivo. We show that such cells accumulate with aging or other models of injury, and that they exhibit clinically targetable features of cellular senescence.

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16INK4a, p19ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.

SummaryCellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro‐inflammatory molecules known as the senescence‐associated secretory phenotype (SASP). The senescence biomarker p16 INK 4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16 INK 4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16 INK 4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16 INK 4a in murine and human models of chondrocyte aging. We observed that p16 INK 4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50‐fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r 2 = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin‐dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16 INK 4a expression. Moreover, increased chondrocyte p16 INK 4a expression correlated with several SASP transcripts. Despite the relationship between p16 INK 4a expression and these features of senescence, somatic inactivation of p16 INK 4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16 INK 4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

Cellular senescence is a phenotypic state that contributes to age‐related diseases through the secretion of matrix‐degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using p16Ink4a expression as a biomarker of senescence. Growth‐factor stimulation of explants increased the expression of p16Ink4a at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16‐high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16‐high cells showed higher senescence‐associated β‐galactosidase activity and increased gene expression of the senescence‐associated secretory phenotype as compared with p16‐low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT‐263). Navitoclax treatment reduced the percentage of p16‐high cells from 17.9 to 6.1% (mean of 13 matched pairs; P < 0.001) and increased cleaved caspase‐3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.—Sessions, G. A., Copp, M. E., Liu, J.‐Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16INK4a‐expressing chondrocytes in cartilage explant culture. FASEB J. 33, 12364–12373 (2019). http://www.fasebj.org