✍🏼 Kotb Abdelmohsen, Krystyna Mazan-Mamczarz, Rachel Munk, Dimitrios Tsitsipatis, Qiong Meng, Martina Rossi, Apala Pal, Chang Hoon Shin, Jennifer L. Martindale, Yulan Piao, Jinshui Fan, Hagai Yanai, Supriyo De, Isabel Beerman, Myriam Gorospe

🏠 Laboratory of Genetics and Genomics, National Institutes of Health (NIH), Baltimore, Maryland, USA

📑 Aging Cell (2024)

Abstract
Cellular senescence, a state of persistent growth arrest, is closely associated with aging and age-related diseases. Deciphering the heterogeneity within senescent cell populations and identifying therapeutic targets are paramount for mitigating senescence-associated pathologies. In this study, proteins on the surface of cells rendered senescent by replicative exhaustion and by exposure to ionizing radiation (IR) were identified using mass spectrometry analysis, and a subset of them was further studied using single-cell CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis. Based on the presence of proteins on the cell surface, we identified two distinct IR-induced senescent cell populations: one characterized by high levels of CD109 and CD112 (cluster 3), the other characterized by high levels of CD112, CD26, CD73, HLA-ABC, CD54, CD49A, and CD44 (cluster 0). We further found that cluster 0 represented proliferating and senescent cells in the G1 phase of the division cycle, and CITE-seq detection of cell surface proteins selectively discerned those in the senescence group. Our study highlights the heterogeneity of senescent cells and underscores the value of cell surface proteins as tools for distinguishing senescent cell programs and subclasses, paving the way for targeted therapeutic strategies in disorders exacerbated by senescence.

How the WOLF was used in this study
The WOLF G2 cell sorter was used as a critical preparative step for high-resolution single-cell analysis of senescent fibroblasts. After WI-38 cells were labeled with barcoded TotalSeq™ antibodies targeting senescence-associated surface markers and stained with propidium iodide to exclude dead cells, the WOLF G2 was employed to sort viable, antibody-labeled cells with high precision. A defined population of 16,000 live cells was isolated to ensure sample purity and optimal cell quality prior to downstream CITE-seq and single-cell RNA sequencing using the 10× Genomics platform. This sorting step enabled accurate coupling of cell surface protein expression with transcriptomic profiles, supporting the study’s goal of resolving heterogeneity among proliferating and senescent cell subpopulations at single-cell resolution.