Senescence profiling and biomarker identification in cell product based on adipose tissue-derived mesenchymal stromal...

Adipose tissue-derived mesenchymal stromal cells (ASC) are used in advanced therapy medicinal products due to their regenerative and immunomodulatory properties. Increasing the number of dosages derived from each donor product is essential to reduce variability and improve scalability of cell therapy. However, extended in vitro expansion may induce cellular senescence, potentially compromising therapeutic efficacy. This study aimed to assess the remaining proliferative potential of a cryopreserved ASC product and identify robust transcriptomic -biomarkers of senescence.

ASC from five donors were cultured until replicative senescence or passage 10. Morphology, growth kinetics, and confluence were monitored. Bulk RNA sequencing was performed on samples from passage 1, 3, 6, and final passage. Principal component analysis, differential expression, gene set variation analysis, and variance partitioning were used to characterize transcriptional changes and identify biomarkers.

ASC maintained stable proliferation and morphology for at least three passages post-thaw. Major transcriptional shifts occurred between passage 3 and later passages. Senescence-associated gene enrichment increased progressively, with donor-specific variation evident at intermediate passages. Forty biomarkers (20 upregulated, 20 downregulated) were identified with expression changes primarily attributable to passage rather than donor identity.

Cryopreserved ASC retain substantial proliferative capacity post-thaw. Senescence develops gradually and is detectable through consistent transcriptomic changes. These findings relate to proliferative and senescence-associated molecular changes and do not directly assess therapeutic efficacy. The identified biomarkers provide a foundation for developing senescence-focused quality control assays to support safe and effective ASC-based therapies.Significance statementThis study identifies promising biomarker candidates for developing a quality control assay to detect cellular senescence and evaluates the remaining replicative potential of a GMP-approved investigational medicinal product. These findings contribute to improving the safety and consistency of cell-based therapies by enabling detection of senescence-related changes.