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Abstract：
BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in the development of therapeutic tools in regenerative medicine. However, their quality decreases during in vitro expansion because of heterogeneity and acquired cellular senescence. We investigated the potential role of podoplanin (PDPN) in minimizing cellular senescence and maintaining the stemness of tonsil-derived MSCs (TMSCs).
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.
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.
摘要：
背景：间充质干细胞（MSC）广泛用于再生医学治疗工具的开发。然而,由于异质性和获得性细胞衰老，它们的质量在体外扩增期间降低。我们研究了足planin（PDPN）在最小化细胞衰老和维持扁桃体来源的MSCs（TMSCs）的干性方面的潜在作用。
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。
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。
