METHODS: TMSCs were isolated from human tonsil tissues using an enzymatic method, expanded, and divided into two groups: early-passaged TMSCs, which were cultured for 3-7 passages, and late-passaged TMSCs, which were passaged more than 15 times. The TMSCs were evaluated for cellular senescence and MSC characteristics, and PDPN-positive and -negative cells were identified by fluorescence-activated cell sorting. In addition, MSC features were assessed in siRNA-mediated PDPN-depleted TMSCs.
RESULTS: TMSCs, when passaged more than 15 times and becoming senescent, exhibited reduced proliferative rates, telomere length, pluripotency marker (NANOG, OCT4, and SOX2) expression, and tri-lineage differentiation potential (adipogenesis, chondrogenesis, or osteogenesis) compared to cells passaged less than five times. Furthermore, PDPN protein levels significantly decreased in a passage-dependent manner. PDPN-positive cells maintained their stemness characteristics, such as MSC-specific surface antigen (CD14, CD34, CD45, CD73, CD90, and CD105) and pluripotency marker expression, and exhibited higher tri-lineage differentiation potential than PDPN-negative cells. SiRNA-mediated silencing of PDPN led to decreased cell-cycle progression, proliferation, and migration, indicating the significance of PDPN as a preliminary senescence-related factor. These reductions directly contributed to the induction of cellular senescence via p16Ink4a/Rb pathway activation.
CONCLUSIONS: PDPN may serve as a novel biomarker to mitigate cellular senescence in the clinical application of MSCs.
方法:使用酶法从人扁桃体组织中分离TMSCs,展开,分为两组:早期传代的TMSCs,培养了3-7个传代,和晚期传代的TMSCs,超过15次。评估TMSCs的细胞衰老和MSC特征,通过荧光激活细胞分选鉴定PDPN阳性和阴性细胞。此外,在siRNA介导的PDPN耗尽的TMSC中评估MSC特征。
结果:TMSCs,当传代超过15次并变得衰老时,表现出降低的增殖率,端粒长度,多能性标记(NANOG,OCT4和SOX2)表达,和三谱系分化潜力(脂肪生成,软骨形成,或成骨)与传代少于五次的细胞相比。此外,PDPN蛋白水平以传代依赖性方式显著降低。PDPN阳性细胞保持其干性特征,例如MSC特异性表面抗原(CD14,CD34,CD45,CD73,CD90和CD105)和多能性标记表达,并显示出比PDPN阴性细胞更高的三谱系分化潜力。SiRNA介导的PDPN沉默导致细胞周期进程减少,扩散,和移民,表明PDPN作为一个初步的衰老相关因子的意义。这些减少直接有助于通过p16Ink4a/Rb途径激活诱导细胞衰老。
结论:PDPN在MSCs的临床应用中可能作为一种新的生物标志物来减轻细胞衰老。