Circular RNA Encoding Yamanaka Factors Enables Partial Reprogramming Without Pluripotency
At the high level, exposing a cell to the Yamanaka factors first does nothing, then later rejuvenates epigenetic control over nuclear DNA structure and gene expression, and then later still causes the cell to dedifferentiate into an induced pluripotent stem cell. Much of the challenge in building rejuvenation therapies based on reprogramming via Yamanaka factors lies in ensuring sufficient epigenetic rejuvenation (desired and beneficial) without any dedifferentiation (undesirable and potentially cancerous) in a tissue environment in which different cell types may require different doses and timing of exposure to Yamanaka factors. With that in mind, the research here is an interesting attempt to close the door on dedifferentiation as a possibility. It remains to be seen as to how useful this will be, but any robust way to ensure that reprogramming will remain partial at all doses and durations will be a very valuable technology.
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.