A circRNA platform for safe partial reprogramming and rejuvenation of mesenchymal stem cells without pluripotent conversion

Mesenchymal stem cells (MSCs) are widely used in cell therapies; however, replicative senescence during in vitro expansion limits their potency and consistency. Partial reprogramming has emerged as a promising strategy to rejuvenate aged cells, yet concerns regarding unintended pluripotent conversion have hindered its translational application. Here we developed a circular RNA (circRNA)-based platform enabling safe partial reprogramming and rejuvenation of MSCs. We generated a lipid nanoparticle (LNP)-delivered circRNA co-expressing OCT4, SOX2, and KLF4 (circ-OSK) to achieve transient and coordinated expression of partial reprogramming factors without genomic modification in MSCs. Using a rigorously validated droplet digital PCR (ddPCR) system targeting the pluripotency marker ESRG , we demonstrated that circ-OSK delivery alone is insufficient to transform MSCs into induced pluripotent stem cells (iPSCs). Circ-OSK-mediated partial reprogramming significantly rejuvenated senescent late-passage MSCs by reducing senescence-associated β-galactosidase activity, DNA damage markers, and inflammatory cytokine secretion, while preserving canonical MSC surface markers and genome stability, and restoring proliferative, migratory and tri-lineage differentiation capacities at a level comparable to early-passage MSCs. The rejuvenated MSCs generated through this process are defined as partially reprogrammed MSCs (prMSCs). Further RNA-seq analysis revealed that prMSCs restored youthful gene expression and promoted a stress resilient and regenerative transcriptional program via repressing the PI3K-Akt signaling pathway. Combined RNA-seq and SNP array analysis confirmed circ-OSK-mediated partial reprogramming retained MSC identity and genome integrity without pluripotent conversion. Our study has established a circRNA-mediated partial reprogramming strategy that rejuvenates MSCs without pluripotent conversion, offering a safety-oriented approach to enhance the potency and quality of MSC-based cell therapy products.