Hypothesis

Clearing senescent cells with a senolytic dashatinib‑quercetin (D+Q) regimen before transient OSK expression will increase the efficiency and safety of epigenetic reprogramming by lowering SASP‑driven histone deacetylase (HDAC) activity that otherwise impedes TET‑dependent DNA demethylation.

Mechanistic Rationale

• Senescent cells secrete a senescence‑associated secretory phenotype (SASP) rich in IL‑6, TGF‑β, and PAI‑1, which activate SMAD and NF‑κB signaling pathways in neighboring cells.
• These pathways upregulate class I/II HDACs (e.g., HDAC1, HDAC2) leading to a more compact chromatin state that limits access of OSK factors to target loci and reduces TET enzyme activity.
• Senolytics such as D+Q selectively induce apoptosis in senescent cells, thereby decreasing SASP levels and downstream HDAC expression.
• Lower HDAC activity results in increased histone acetylation, a more open chromatin landscape, and enhanced TET‑mediated 5‑mC oxidation, facilitating the DNA demethylation waves observed during partial reprogramming.

Predictions

1. Primary – In aged mice, a regimen of D+Q followed by a single 1‑week OSKM induction will yield a greater reduction in epigenetic age (measured by Horvath‑style DNA methylation clocks) in liver, pancreas, and spleen compared with OSKM alone or D+Q alone.
2. Secondary – Tissue‑specific functional readouts (e.g., glucose tolerance test for pancreas, ALT/AST levels for liver, serum albumin for spleen) will show superior improvement only in the sequential group.
3. Mechanistic biomarker – HDAC1/2 protein levels and global H3K9ac/H3K27ac marks will be significantly lower in the sequential group than in OSKM‑only controls, correlating with increased 5‑hmC levels.

Experimental Design

• Animals: 20‑month‑old C57BL/6J mice, n=10 per group.
• Groups: (1) Vehicle control, (2) D+Q (dasatinib 5 mg/kg + quercetin 50 mg/kg, 3 consecutive days), (3) OSKM induction (doxycycline‑inducible OSKM, 1 week), (4) Sequential D+Q → OSKM (D+Q administered 3 days before doxycycline induction).
• Readouts:
  • Epigenetic age via reduced representation bisulfite sequencing (RRBS) at baseline and 2 weeks post‑intervention.
  • Western blot / immunostaining for HDAC1/2, H3K9ac, H3K27ac, and 5‑hmC in target tissues.
  • SASP cytokine profiling (IL‑6, TGF‑β) in serum and tissue homogenates.
  • Functional assays as described above.
• Statistical analysis: One‑way ANOVA with Tukey post‑hoc; significance set at p<0.05.

Falsifiability

If the sequential group does not show a statistically significant greater reduction in epigenetic age or functional improvement compared with OSKM alone, and HDAC/acetyl‑marker levels are not differentially altered, the hypothesis would be falsified. Conversely, a confirmed increase in chromatin openness and demethylation only when senolytics precede reprogramming would support the mechanistic link between SASP‑mediated HDAC inhibition and reprogramming efficiency.