Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications, large scale production is required and optimal cryopreservation and culture conditions are essential to autologous and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term culture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological characteristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated. Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology, surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and proteomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continuously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were confirmed by mass spectrometry. The results showed no significant differences in phenotypes including morphology, surface marker and tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell cycle and other pathways. This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs applications in regenerative medicine.

中文翻译：

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冷冻保存和长期培养对人脐带间充质干细胞生物学特性和蛋白质组学特征的影响。

人脐带来源的 MSCs (hUC-MSCs) 已被确定为在组织工程和再生医学临床应用中很有前途的种子细胞，因为它们具有简单的获取程序和来自年轻组织供体的能力，而不是其他 MSCs 来源. 在临床应用中，需要大规模生产，最佳的冷冻保存和培养条件对于未来的自体和同种异体移植至关重要。然而，冷冻保存的解冻后和长期培养对 hUC-MSCs 的影响仍然未知，特别是在特定蛋白质表达方面。因此，研究了冷冻保存和长期培养后 hUC-MSCs 的生物学特性和蛋白质组学特征。首先，从人脐带组织中分离hUC-MSCs，在第3代通过形态学、表面标志物和三向分化潜能进行鉴定，然后在第4代冷冻保存和长期培养后检测并比较其生物学特征和蛋白质组学特征，连续培养至第 10 代，并在此处进行检测。通过使用等压标签进行相对和绝对定量 (iTRAQ) 标记技术测试蛋白质组学特征，并通过质谱法确认差异蛋白质。结果表明，形态学、表面标志物和三系分化潜能等表型无显着差异，但翻译水平有明显变化，涉及代谢、细胞周期等途径。