Senescent fibroblasts retain tissue-specific epigenetic memory that biases iPSC reprogramming toward fibroblastic fate

Hypothesis

Transiently senescent fibroblasts preserve a tissue-specific epigenetic landscape that encodes positional and developmental information. When these cells are cleared by senolytics, this instructive memory is lost, reducing the fidelity of induced pluripotent stem cell (iPSC) reprogramming in the local microenvironment and skewing differentiation away from the original tissue lineage.

Mechanistic basis

Recent work shows that senescent fibroblasts activated during acute wound healing express developmental and morphogenetic gene programs [[https://doi.org/10.1101/2025.06.08.658533]]. These cells secrete SASP factors such as PDGF‑AA that drive myofibroblast recruitment and immune cell activation [[https://pmc.ncbi.nlm.nih.gov/articles/PMC9775319/]]. Beyond paracrine signaling, prolonged residence in a niche imposes stable chromatin marks—e.g., H3K27ac at enhancers of fibroblast‑specific genes and reduced DNA methylation at loci governing extracellular matrix composition. This epigenetic memory persists even after the senescence-associated secretory phenotype wanes, effectively bookmarking the cell’s tissue of origin.

If senescent fibroblasts act as epigenetic chaperones, their removal should diminish the local pool of cells capable of imparting lineage‑biased cues during reprogramming. iPSCs generated in a senolytic‑depleted environment would therefore show reduced expression of fibroblast‑specific markers and increased propensity toward alternative mesenchymal or non‑mesenchymal lineages when subjected to differentiation cues that normally favor fibroblast fate.

Testable predictions

1. Epigenetic profiling – Compare ATAC‑seq and whole‑genome bisulfite sequencing of senescent fibroblasts isolated from young mouse skin before and after ABT‑263 treatment. Predict loss of fibroblast‑specific enhancer accessibility and gain of repressive marks at lineage‑defining loci.
2. Reprogramming efficiency – Transduce equal numbers of senescent and non‑senescent fibroblasts with OSKM factors in vitro, then measure iPSC colony formation and transcriptomic similarity to native fibroblasts. Expect senescent cells to yield iPSCs with higher fibroblast‑signature scores.
3. In vivo lineage tracing – Use a Cre‑loxP system to label senescent fibroblasts (p16‑3MR) in dorsal skin, administer senolytics, then induce localized injury and reprogramming via transient OSKM expression. Quantify the proportion of regenerated fibroblasts derived from labeled precursors versus unlabeled sources. Prediction: senolytic treatment will decrease the contribution of labeled fibroblasts to the regenerated stroma.
4. Functional outcome – Assess wound tensile strength and histology after injury in mice receiving senolytics versus controls. If epigenetic chaperone loss impairs proper fibroblast‑mediated matrix deposition, senolytic‑treated wounds will show abnormal collagen organization despite reduced senescent cell burden.

Falsification

If senolytic clearance does not alter the epigenetic state of remaining fibroblasts, or if iPSCs generated post‑senolysis retain fibroblast‑specific transcriptional programs at rates comparable to untreated controls, the hypothesis that senescent cells serve as epigenetic memory reservoirs would be refuted. Similarly, if lineage‑tracing shows no change in fibroblast contribution to regenerated tissue despite senescent cell depletion, the chaperone role would be unsupported.

Broader implication

This framework reframes senolytics not merely as clearance agents but as potential disruptors of tissue‑specific epigenetic instruction. Therapeutic strategies might need to temporally separate senolytic administration from regenerative windows, or to supplement cleared niches with exogenous epigenetic cues (e.g., fibroblast‑derived extracellular vesicles) to preserve positional information during regeneration.