Exosomal miR-302b rejuvenates aging mice by reversing cell cycle arrest [LO2 scRNA-Seq]

Cellular senescence is a hallmark of aging characterized by a stable exit from the cell cycle in response to cellular damage and stress. Senescent cells (SnCs) are closely associated with aging and aging-related disorders, making them a potential target for slowing aging. In this study, we developed a tool for dynamically monitoring the p21 signaling to determine the degree of cellular senescence, named p21-YFP LO2. The senescent cells were prepared by FCM sorting targeting p21-YFP LO2 treated with 50 nM Dox for 48 h based on the YFP signaling. The single-cell RNA sequencing (scRN-seq) analysis was performed to observe the effects of human embryonic stem cell-derived exosomes (hESC-Exos) on the transcriptional profiles of SnCs. We specifically demonstrated that hESC-Exos enable SnCs to reverse the cell cycle arrest and restore their proliferative capacity in vitro. Overall design: A senescence reporter cell line was generated by inserting the YFP gene downstream of the Cdkn1a (code p21) stop codon and subsequently transfecting LO2 cells, named p21-YFP LO2. Temporal fluctuations in p21 expression were visualized using the YFP signal. A senescent cell model was established by treating the p21-YFP LO2 with 50 nM Dox for 48 h. To observe the effects of hESC-Exos on the transcriptional profiles of SnCs, we performed single-cell RNA sequencing (scRN-seq) on SnCs sorted by FCM and then treated with hESC-Exos or PBS, termed as Dox-pbs and Dox-Exos group, respectively.