Department for Life Quality Studies, Alma Mater Studiorum, University of Bologna, Corso d'Augusto 237, 47921 Rimini, Italy.
School of Pharmacy, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy.
Oxid Med Cell Longev. 2018 Dec 17;2018:5263985. doi: 10.1155/2018/5263985. eCollection 2018.
Amniotic fluid stem cells (AFSCs) are characterized by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation . SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.
羊水干细胞（AFSCs）的独特生态位特征保证了其在体内的稳态作用。为了将干细胞的功能应用于临床，需要不断改进细胞培养条件。细胞氧化还原状态在干细胞生物学中起着重要作用，只要活性氧（ROS）浓度得到精细调节并排除其不利影响。本研究旨在研究两种抗氧化剂——萝卜硫素（SF）和表没食子儿茶素没食子酸酯（EGCG）——对 AFSCs 因高氧和冻融循环引起的氧化应激的保护作用。从健康受试者中分离和鉴定人羊水干细胞。进行代谢功能和抗氧化活性测定，以研究 SF 和 EGCG 共同处理对 AFSCs 的影响。实时 PCR 用于研究共同处理对多能性、衰老、成骨和成脂标志物和抗氧化酶的影响。碱性磷酸酶测定和茜素红染色用于确认成骨分化。SF 和 EGCG 的共同处理可有效减少 ROS 产生，增加 GSH 水平，并通过上调谷胱甘肽还原酶、NAD（P）H：醌氧化还原酶-1 和硫氧还蛋白还原酶来增强内源性抗氧化防御。有趣的是，共同处理通过上调多能性标志物如 OCT4 和 NANOG 来维持干细胞状态。此外，共同处理影响未处理细胞的衰老相关基因标志物。共同处理上调成骨基因标志物并促进成骨分化。SF 和 EGCG 可在 AFSC 培养中联合使用，作为维持干细胞功能的策略。
