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Background: Adipose-derived stem cells (ADSCs) have been extensively used in preclinical and clinical trials for treating various diseases. However, the differences between ADSCs from lean individuals (L-ADSCs) and those from obese individuals (O-ADSCs) have not been thoroughly investigated, particularly regarding their mitochondrial and lysosomal functions. Therefore, this study aims to evaluate the differences between L-ADSCs and O-ADSCs in terms of cell biological activity, mitochondria, and lysosomes., Methods: We first isolated and cultured L-ADSCs and O-ADSCs. We then compared the differences between the two groups in terms of biological activity, including cell proliferation, differentiation potential, and their effect on the polarization of macrophages. Additionally, we observed the mitochondrial and lysosomal morphology of ADSCs using an electronic microscope, MitoTracker Red, and lysotracker Red dyes. We assessed mitochondrial function by examining mitochondrial membrane potential and membrane fluidity, antioxidative ability, and cell energy metabolism. Lysosomal function was evaluated by measuring autophagy and phagocytosis. Finally, we performed transcriptome analysis of the ADSCs using RNA sequencing., Results: The biological activities of O-ADSCs were decreased, including cell immunophenotypic profiles, cell proliferation, and differentiation potential. Furthermore, compared to L-ADSCs, O-ADSCs promoted M1-type macrophage polarization and inhibited M2-type macrophage polarization. Additionally, the mitochondrial morphology of O-ADSCs was altered, with the size of the cells becoming smaller and mitochondrial fragments increasing. O-ADSCs also exhibited decreased mitochondrial membrane potential and membrane fluidity, antioxidative ability, and energy metabolism. With respect to lysosomes, O-ADSCs contained ungraded materials in their lysosomes, enhanced lysosomal permeability, and reduced autophagy and phagocytosis ability. RNA sequence analysis indicated that the signalling pathways related to cell senescence, cancer, and inflammation were upregulated, whereas the signalling pathways associated with stemness, cell differentiation, metabolism, and response to stress and stimuli were downregulated., Conclusions: This study indicates that ADSCs from individuals (BMI > 30 kg/m 2 ) exhibit impaired mitochondrial and lysosomal function with decreased biological activity., (© 2024. The Author(s).)

Background: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]