Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton’s jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

摘要 目的：本研究旨在采用相较于金标准蛋白质免疫印迹（Western blotting）技术更实用、快速且低成本的方法，以经典衰老标志物p16（INK4A）和p21（CDKN1A）追踪脐带间充质干细胞的扩增过程，并评估该方法在实验室中的应用可行性。方法：从沃顿胶（Wharton’s jelly）中分离脐带间充质干细胞，经质量控制、形态学及流式细胞术免疫表型鉴定后，进行体外培养扩增直至接近细胞周期阻滞（复制性衰老）阶段。结果：选取第5代年轻细胞与第10代预衰老细胞进行对比，评估经典细胞衰老标志物p16与p21的蛋白表达水平，将蛋白质免疫印迹（Western blotting）的检测结果与流式细胞术、间接免疫荧光法的所得结果进行比较。结论：通过间接免疫荧光法检测p16的表达来追踪细胞培养状态，可识别出脐带间充质干细胞培养物中存在复制性衰老风险的细胞群体。