Dental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs. DPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0–200 μM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-β-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays. High proliferative DPSCs underwent > 80PDs in culture and resisted H2O2−induced senescence (50–76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4–32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2–10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45–60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10–25PDs), which declined during expansion. Low proliferative DPSCs (2–10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities. Significant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications.

中文翻译：

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SOD2和GSTZ1谱的差异有助于对比牙髓干细胞对氧化损伤和早衰的敏感性

牙髓干细胞（DPSC）越来越多地被提倡为基于再生医学的疗法的可行细胞来源。然而，DPSC扩展和多潜能的显着异质性是公认的，这归因于端粒轮廓和对复制衰老的敏感性。由于DPSC的人类端粒酶（hTERT）表达可忽略不计，我们通过DPSC之间的酶抗氧化能力差异，研究了DPSC亚群对氧化应激诱导的生物分子损伤和早衰敏感性的内在差异是否进一步促成了这种异质性。通过差异纤连蛋白的粘附力从人的第三磨牙中分离出DPSC，证实了间充质细胞（CD73 / CD90 / CD105）阳性和造血细胞（CD45）阴性表达。分离的亚群在H2O2（0–200μM）中扩增，并根据种群倍增（PD）和衰老（端粒长度，SA-β-半乳糖苷酶，p53 / p16INK4a / p21waf1 / hTERT）建立为高或低增殖DPSC。标记检测。评估了DPSC扩增对间充质，胚胎和神经rest标志物表达的影响，以及通过免疫细胞化学对高和低增殖DPSC对氧化DNA和蛋白质损伤的敏感性。通过qRT-PCR，Western印迹和活性分析进一步比较了DPSC亚群之间的超氧化物歧化酶（SODs），过氧化氢酶和谷胱甘肽相关的抗氧化剂的表达谱。高增殖DPSC经历了> 培养物中的80PDs和抗H2O2诱导的衰老（50-76PDs）。相反，即使在未经治疗的对照组（11-34PDs）中，低增殖亚群也显示出加速衰老（4-32PDs）。尽管端粒长度在很大程度上不受影响，但某些干细胞标记物的表达随H2O2处理和扩增而下降。低增殖DPSCs（2-10PDs）的衰老敏感性增加，伴随着氧化损伤的增加，高增殖DPSCs直到45-60PDs才存在。在高增殖DPSC（10–25PDs）中，SOD2 /谷胱甘肽S-转移酶ζ1（GSTZ1）的表达和SOD活性增加，在扩张过程中下降。低增殖DPSC（2-10PDs）的SOD，过氧化氢酶和谷胱甘肽相关的抗氧化剂表达/活性较差。DPSC亚群对氧化损伤和过早衰老的敏感性存在显着差异，这归因于在高增殖DPSC中维持抗衰老/干性特性的SOD2和GSTZ1谱的差异。鉴定高增殖DPSC中优异的抗氧化剂特性可增强我们对DPSC生物学和衰老的了解，可将其用于从牙髓组织中进行选择性亚群筛选/分离，以用于基于再生医学的应用。