Isolation and identification of senescent renal tubular epithelial cells using immunomagnetic beads based on DcR2


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Abstract

Cell senescence plays a major role in the progression of tumors and chronic conditions such as diabetes and chronic kidney disease. Senescent cells are an important model for the study of aging-related diseases, and there is currently no efficient method for sorting out senescent cells. Decoy receptor 2 (DcR2) is a transmembrane receptor of the tumor necrosis factor superfamily, which is specifically expressed in senescent cells. In this study, we used magnetic activated cell sorting (MACS) isolation of a highly-pure populations DcR2-positive renal tubular epithelial cells (RTECs) based on three senescent cell models including the fifth passage cells, advanced glycation end-products (AGEs)- and H2O2-induced cells. The percentages of DcR2 positive RTECs in G1 and S phases increased by 20% and 4%, respectively, as compared to that in the pre-sorted cells. The positivity rates of SA-β-gal, p16, and senescence-associated heterochromatin foci (SAHF) in DcR2-positive RTECs were about 40%, 30%, and 44% higher than that prior to cell sorting. The levels of IL-6 and TGF-β1 in the supernatant were increased by 1.7 and 1.5 folds, respectively, as compared to that observed prior to sorting. No significant cell death was observed after 5 days of continuous culture. Ki-67 positive expression rate in DcR2 negative RTECs was significantly higher than that in DcR2 positive RTECs after MACS. We demonstrated the use of DcR2 to classify live, senescent RTECs with a high specificity and stability. Our findings lay the foundation for further study of senescent RTECs in the progression of chronic kidney disease.HighlightsThis study demonstrated for the first time DcR2 can be used to isolate and purify senescent RTECs with a high specificity and stability in three classical senescent models with MACS.The purified live senescent RTECs were successfully sort out, which lay the foundation for further study of senescent RTECs in the progression of kidney diseases.

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