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

细胞衰老（cellular senescence）是衰老的标志性特征，其特征为细胞在遭遇损伤与应激后发生稳定的细胞周期退出。衰老细胞（Senescent Cells, SnCs）与衰老及衰老相关疾病密切相关，故而成为延缓衰老的潜在干预靶点。本研究开发了一款可动态监测p21信号通路以量化细胞衰老程度的工具，命名为p21-YFP LO2。研究人员以50 nM多柔比星（Doxorubicin, Dox）处理p21-YFP LO2细胞48小时，基于黄色荧光蛋白（YFP）信号通过流式细胞术（FCM）分选获得衰老细胞。本研究通过单细胞RNA测序（single-cell RNA sequencing, scRN-seq）分析人胚胎干细胞衍生外泌体（hESC-Exos, human embryonic stem cell-derived exosomes）对衰老细胞转录组谱的影响，并明确证实：人胚胎干细胞衍生外泌体可在体外使衰老细胞逆转细胞周期阻滞，恢复其增殖能力。
总体实验设计：研究人员将黄色荧光蛋白（YFP）基因插入至Cdkn1a（编码p21）终止密码子下游，随后转染LO2细胞，构建得到衰老报告细胞系，命名为p21-YFP LO2，通过YFP信号可可视化p21表达的时序波动。研究人员以50 nM多柔比星（Dox）处理p21-YFP LO2细胞48小时，建立衰老细胞模型。为探究hESC-Exos对衰老细胞转录组谱的影响，本研究对经FCM分选的衰老细胞分别施以hESC-Exos或磷酸盐缓冲液（Phosphate Buffered Saline, PBS）处理，随后进行单细胞RNA测序（scRN-seq），其中PBS处理组命名为Dox-pbs组，外泌体处理组命名为Dox-Exos组。