State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, P.O. Box 329#, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
State Key Laboratory of Oncogenes and Related Genes, Renji-MedX Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China.
Stem Cell Res Ther. 2022 Aug 13;13(1):411. doi: 10.1186/s13287-022-03107-4.
Mesenchymal stem cells (MSCs) have been extensively used for the treatment of various diseases in preclinical and clinical trials. In vitro propagation is needed to attain enough cells for clinical use. However, cell aging and viability reduction caused by long-time culture have not been thoroughly investigated, especially for the function of mitochondria and lysosomes. Therefore, this study was designed to detect mitochondrial and lysosomal activity, morphological and functional changes in human umbilical cord MSCs (UMSCs) after long-time culture.
First, we examined cell activities, including proliferation and immigration ability, differentiation potential, and immunosuppressive capacity of UMSCs at an early and late passages as P4 (named UMSC-P4) and P9 (named UMSC-P9), respectively. Then, we compared the mitochondrial morphology of UMSC-P4 and UMSC-P9 using the electronic microscope and MitoTracker Red dyes. Furthermore, we investigated mitochondrial function, including mitochondrial membrane potential, antioxidative ability, apoptosis, and ferroptosis detected by respective probe. Cell energy metabolism was tested by mass spectrometry. In addition, we compared the lysosomal morphology of UMSC-P4 and UMSC-P9 by electronic microscope and lysoTracker Red dyes. Finally, the transcriptome sequence was performed to analyze the total gene expression of these cells.
It was found that UMSC-P9 exhibited a reduced biological activity and showed an impaired mitochondrial morphology with disordered structure, reduced mitochondrial crista, and mitochondrial fragments. They also displayed decreased mitochondrial membrane potential, antioxidative ability, tricarboxylic acid cycle activity and energy production. At the same time, apoptosis and ferroptosis were increased. In addition, UMSC-P9, relative to UMSC-P4, showed undegraded materials in their lysosomes, the enhancement in lysosomal membrane permeability, the reduction in autophagy and phagocytosis. Moreover, transcriptome sequence analysis also revealed a reduction of cell function, metabolism, mitochondrial biogenesis, DNA replication and repair, and an increase of gene expression related to cell senescence, cancer, diseases, and infection in UMSC-P9.
This study indicates that in vitro long-time culturing of MSCs can cause mitochondrial and lysosomal dysfunction, probably contributing to the decline of cell activity and cell aging. Therefore, the morphology and function of mitochondria and lysosomes can be regarded as two important parameters to monitor cell viability, and they can also serve as two important indicators for optimizing in vitro culture conditions.
间充质干细胞（MSCs）已广泛应用于临床前和临床试验中各种疾病的治疗。为了达到临床应用所需的足够细胞数量，需要进行体外扩增。然而，长时间培养导致的细胞衰老和活力降低问题尚未得到彻底研究，尤其是线粒体和溶酶体的功能。因此，本研究旨在检测人脐带间充质干细胞（UMSC）在长时间培养后线粒体和溶酶体的活性、形态和功能变化。
首先，我们分别检测了早期和晚期传代的 UMSC（命名为 UMSC-P4 和 UMSC-P9）的细胞活性，包括增殖和迁移能力、分化潜能和免疫抑制能力。然后，我们使用电子显微镜和 MitoTracker Red 染料比较了 UMSC-P4 和 UMSC-P9 的线粒体形态。此外，我们通过相应的探针检测了线粒体功能，包括线粒体膜电位、抗氧化能力、细胞凋亡和铁死亡。通过质谱法检测细胞能量代谢。此外，我们通过电子显微镜和 lysoTracker Red 染料比较了 UMSC-P4 和 UMSC-P9 的溶酶体形态。最后，进行了转录组序列分析以分析这些细胞的总基因表达。
研究发现，UMSC-P9 的生物活性降低，线粒体形态结构紊乱，线粒体嵴减少，线粒体片段化。同时，线粒体膜电位、抗氧化能力、三羧酸循环活性和能量产生也降低，细胞凋亡和铁死亡增加。此外，UMSC-P9 的溶酶体中未降解物质增多，溶酶体膜通透性增强，自噬和吞噬作用减少。此外，转录组序列分析还表明，UMSC-P9 中与细胞功能、代谢、线粒体生物发生、DNA 复制和修复相关的基因表达减少，而与细胞衰老、癌症、疾病和感染相关的基因表达增加。
本研究表明，MSCs 体外长时间培养可导致线粒体和溶酶体功能障碍，可能导致细胞活性和细胞衰老下降。因此，线粒体和溶酶体的形态和功能可以作为监测细胞活力的两个重要参数，也可以作为优化体外培养条件的两个重要指标。
