Crotonylation Boost Keeps Aging Stem Cells Fighting Gut Inflammation

Stem cell therapies for inflammatory bowel disease have long been held back by a stubborn problem: the cells themselves age. Now a research team reports that a chemical tag called lysine crotonylation declines as adipose-derived mesenchymal stem cells (ADSCs) grow old in culture, and that restoring this modification with a simple metabolite can rejuvenate the cells and sharpen their ability to calm inflamed intestines in mice. The study, published in Cell Regeneration, traces the effect to an enzyme named ACSS2 and describes a self-reinforcing epigenetic loop that keeps the cells young and anti-inflammatory.

Inflammatory bowel disease, which includes Crohn’s disease and ulcerative colitis, involves persistent and recurrent intestinal inflammation and has been rising in prevalence and incidence worldwide. Current treatments, from small-molecule drugs to biological agents, still leave patients without definitive cures. Mesenchymal stem cells have attracted attention as a potential therapy because of their anti-inflammatory properties and biological safety, and adipose tissue is a particularly convenient source: ADSCs are easy to obtain, show strong anti-inflammatory activity, and have performed well in colitis models. Yet their therapeutic capacity is frequently undermined by cellular senescence that develops during the in vitro expansion needed to produce clinically useful numbers of cells.

The researchers began by comparing ADSCs at passage three with cells at passage seven, an extended stretch of laboratory culture. The older cells showed markedly higher levels of classic senescence markers, including the tumor-suppressor proteins p53, p21 and p16, and stained positive for senescence-associated beta-galactosidase, a standard indicator of aged cells. Alongside this senescent shift, the team documented two parallel declines: global lysine crotonylation levels, measured by immunoblotting with a pan-crotonylation antibody, and intracellular crotonyl-CoA, the small-molecule substrate that enzymes attach to lysine residues, quantified by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Crotonylation, a relatively recently identified post-translational modification, is known to influence proliferation and differentiation in embryonic, periodontal ligament and neural stem cells, but its role in stem cell aging had remained largely unexplored.

To test whether the decline mattered, the researchers supplemented the culture medium with sodium crotonate, a precursor that cells can convert into crotonyl-CoA. At a concentration of 10 millimoles per liter, the treatment significantly reduced senescence markers, lowered senescence-associated beta-galactosidase staining and dampened the senescence-associated secretory phenotype, the inflammatory cocktail of signaling molecules that aged cells release. It also markedly enhanced the cells’ proliferative capacity in growth-curve assays. In other words, feeding the crotonylation pathway appeared to push the aging cells back toward a younger, more vigorous state.

The critical question was whether this laboratory rejuvenation would translate into better therapy. The team induced colitis in mice with dextran sulfate sodium, a chemical that damages the intestinal lining, and then injected the animals intraperitoneally with one million ADSCs that had been pre-treated with sodium crotonate for 48 hours. Compared with mice receiving untreated control ADSCs, the animals given the pre-treated cells lost less weight, retained longer colons, scored lower on the disease activity index and showed better histopathological scores in stained intestinal tissue. The pre-treatment, the authors conclude, can enhance the anti-colitic efficacy of ADSCs in vivo.

Searching for the mechanism behind the falling crotonylation levels, the researchers performed RNA sequencing on young and senescent ADSCs and examined the genes that govern crotonylation homeostasis: the writer enzymes that add the tag, the eraser enzymes that remove it, and the enzymes that synthesize crotonyl-CoA. One name stood out. ACSS2, short for acyl-CoA short-chain synthetase 2, an enzyme central to crotonyl-CoA biosynthesis, was significantly downregulated in the senescent cells, a finding the team confirmed at both the messenger RNA and protein levels. When the researchers engineered ADSCs to overexpress ACSS2, pan-crotonylation rose, senescence markers and beta-galactosidase staining fell, the senescence-associated secretory phenotype quieted, and proliferation improved.

The dependency ran in the other direction as well. When the team knocked down ACSS2 with short hairpin RNA, pan-crotonylation dropped, senescence markers and secretory-phenotype expression climbed, and proliferation faltered. Crucially, adding sodium crotonate to these ACSS2-deficient cells failed to rescue any of those measures. The anti-senescence effect of the metabolite, the authors write, depends on ACSS2-mediated crotonylation. The same dependency appeared in the colitis model: ACSS2 knockdown not only weakened the inherent anti-colitis activity of the ADSCs but also abolished the extra benefit that sodium crotonate pre-treatment normally confers, with the mice showing more weight loss, shorter colons, higher disease activity scores and worse histological damage.

The study also uncovered a feedback loop that explains how sodium crotonate raises ACSS2 in the first place. Crotonylation of histone H3 at lysine 9, a mark known to facilitate gene transcription, declined as ADSCs aged, and chromatin immunoprecipitation sequencing showed that this mark was significantly reduced at the ACSS2 promoter region in senescent cells, a result validated by chromatin immunoprecipitation followed by quantitative PCR. When the cells were treated with sodium crotonate, the enrichment of H3K9 crotonylation at the ACSS2 gene locus increased, ACSS2 expression rose, and the elevated ACSS2 in turn drove more crotonylation throughout the cell. The result is a positive feedback loop: more crotonyl-CoA substrate leads to more histone marking at the ACSS2 promoter, which produces more ACSS2 enzyme, which generates still more crotonylation.

Finally, the researchers connected the pathway to NF-κB, a classical signaling pathway that promotes cellular senescence. Overexpressing ACSS2 suppressed the phosphorylation and nuclear translocation of p65, the pathway’s central relay, and sodium crotonate treatment produced the same inhibition. In ACSS2-knockdown cells, however, the effects of sodium crotonate were abolished. The authors note that their study did not explore exactly how crotonylation regulates the NF-κB pathway, leaving that question for future work. They also flag other limitations: the investigation concentrated on the ACSS2-H3K9 crotonylation axis, other histone crotonylation marks may contribute to ADSC senescence, whether sodium crotonate acts similarly in other stem cell types is unknown, and the safety profile and long-term effects of pre-conditioned cells still require thorough evaluation in preclinical studies.

Even with those caveats, the researchers argue that the strategy fits naturally into existing cell manufacturing. Numerous pre-treatment approaches, from inflammatory cytokine priming to hypoxic culture, have been documented to boost the anti-inflammatory function and survival of ADSCs, and sodium crotonate pre-treatment is a simple metabolic addition that could be readily integrated into current protocols. The ADSCs used in the study were isolated from surgical specimens of patients with inflammatory bowel disease, authenticated by cytometry after the third passage, and used at early passages for the experiments, with sequencing data deposited in public repositories. By linking metabolism, epigenetic modification and cellular aging in a single pathway, the work identifies lysine crotonylation as a potential therapeutic target and offers a practical route to keeping stem cell therapies potent from the culture flask to the clinic.

Subject of Research: The role of ACSS2-mediated lysine crotonylation in the senescence of adipose-derived stem cells and its effect on stem cell therapy for inflammatory bowel disease

Article Title: ACSS2-mediated lysine crotonylation attenuates senescence and enhances the therapeutic efficacy of adipose-derived stem cells in inflammatory bowel disease

Article References: Yuan, M., Li, S., Chen, S., Zhu, M., Yu, R., Huang, J., Liang, G., Zhang, C., He, X., Lan, P., & Wu, X. (2026). ACSS2-mediated lysine crotonylation attenuates senescence and enhances the therapeutic efficacy of adipose-derived stem cells in inflammatory bowel disease. Cell Regeneration, 15(1), Article 11. https://doi.org/10.1186/s13619-026-00285-x

Image Credits: AI Generated

DOI: 10.1186/s13619-026-00285-x

Keywords: inflammatory bowel disease, adipose-derived stem cells, lysine crotonylation, ACSS2, cellular senescence, crotonyl-CoA, H3K9 crotonylation, NF-κB pathway, sodium crotonate, epigenetics, colitis mouse model, mesenchymal stem cells