Hypothesis: In the aged limbal epithelium, a subpopulation of senescent stromal and epithelial cells secretes an IL‑6‑rich SASP that activates STAT3 signaling in neighboring label‑retaining limbal epithelial stem cells (LESCs), thereby maintaining PAX6 transcription and preserving stem‑cell potency. Complete senolytic clearance of these cells removes this paracrine support, leading to diminished PAX6+ LESC numbers, reduced clonal regeneration, and compromised corneal transparency in native aging. This hypothesis is testable and falsifiable using aged mouse models and lineage‑tracing approaches.

Mechanistic rationale: Recent work shows that transient SASP factors such as IL‑6 can promote stem‑cell self‑renewal through STAT3‑dependent transcription (see [2]). PAX6 expression is essential for LESC differentiation and corneal homeostasis ([4],[5]), yet its regulation by niche signals remains unexplored. We propose that senescent limbal cells, identified by p16^INK4a^ expression and SA‑β‑gal activity, produce IL‑6 and possibly exosomal miR‑205, which together activate STAT3 in adjacent LESCs. STAT3 phosphorylation drives direct binding to PAX6 enhancer regions, sustaining PAX6 mRNA and protein levels. In young tissue, this signal is transient; in aged tissue, senescent cells persist and become a chronic source of supportive IL‑6, creating a paradox where their removal harms regeneration.

Experimental plan: 1) Use p16-3MR mice to label senescent cells in limbal tissue of 18‑month‑old animals. 2) Treat cohorts with either the senolytic ABT‑263 (navitoclax) or a senomorphic agent (ruxolitinib, a JAK1/2 inhibitor) for two weeks. 3) Quantify senescent cell clearance (SA‑β‑gal, p16) and SASP composition (IL‑6 ELISA, multiplex). 4) Assess LESC frequency via label‑retention assays and flow cytometry for ABCG2^−/p63^hi^ cells, and measure PAX6+ LESC proportion by immunofluorescence. 5) Evaluate functional regeneration after epithelial wounding (central corneal epithelial defect) by tracking closure rate and corneal clarity (slit‑lamp scoring). 6) Perform rescue experiments: add recombinant IL‑6 or activate STAT3 with a selective agonist in senolytic‑treated eyes to test whether PAX6+ LESC numbers and healing are restored.

Predictions: If the hypothesis holds, ABT‑263 will reduce senescent cells but also lower IL‑6 levels, decrease STAT3 phosphorylation in LESCs, and cause a significant drop in PAX6+ LESC frequency compared with untreated aged controls. Consequently, wound closure will be delayed and corneal haze increased. Ruxolitinib, which blocks STAT3 without eliminating senescent cells, should phenocopy the senolytic effect on PAX6+ LESCs and healing, confirming that the IL‑6/STAT3 axis—not cell removal per se—drives the outcome. Exogenous IL‑6 or STAT3 activation should rescue PAX6 expression and regenerative capacity despite senolytic treatment. Conversely, if senolytic treatment improves PAX6+ LESC numbers and healing in aged corneas, the hypothesis would be falsified, indicating that senescent cells are detrimental even in native aging.

This study directly addresses the gap identified in the literature—lack of senolytic testing in native aged corneal regeneration—and provides a mechanistic link between senescence‑associated paracrine signaling and the master regulator PAX6. It also predicts that senomorphic strategies may preserve beneficial niche functions while mitigating pathological SASP, offering a refined therapeutic perspective for age‑related ocular surface decline.