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Diabetic sera disrupted the normal exosome signaling pathway in human mesenchymal stem cells in vitro
Jafar Rezaie1, Vahid Nejati2, Majid Khaksar3
1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract:
Human mesenchymal stem cells were exposed to diabetic sera for 7 days. Cell viability and apoptosis rate were detected by MTT and flow cytometry assays. The expression of key genes such as CD63, Alix, Rab27a, Rab27b, and Rab8b was monitored by real-time PCR. We also measured acetylcholinesterase activity and size and zeta potential of exosomes in the supernatant form diabetic cells and control. The cellular distribution of CD63 was shown by immunofluorescence imaging and western blotting. Any changes in the ultrastructure of cells were visualized by electron microscopy. Data showed a slight decrease in survival rate and an increased apoptosis in diabetic cells as compared to control (p < 0.05). By exposing cells to diabetic sera, a significant increase in the level of all genes CD63, Alix, Rab27a, Rab27b, and Rab8b was observed (p < 0.05). Flow cytometry analysis and immunofluorescence imaging confirmed increasing CD63 protein content upon treatment with diabetic sera (p < 0.05). We found an enhanced acetylcholinesterase activity in a diabetic condition which coincided with the increasing size of exosomes and decrease in zeta potential (p < 0.05). The fatty acid profile was not significantly affected by diabetic sera. Ultrastructural examination detected more accumulated cytoplasmic lipid vacuoles in diabetic cells.
Insights
Diabetic sera negatively impact human mesenchymal stem cells, increasing apoptosis and altering exosome characteristics. Key exosome-related gene and protein expression significantly increased in diabetic conditions.
Area of Science:
- Cell Biology
- Stem Cell Research
- Biochemistry
Background:
- Diabetes mellitus is a metabolic disorder with significant cellular consequences.
- Mesenchymal stem cells (MSCs) are crucial for tissue repair and regeneration.
- Diabetic conditions may alter MSC function and their secreted extracellular vesicles (exosomes).
Purpose of the Study:
- To investigate the effects of diabetic sera on human mesenchymal stem cells.
- To analyze changes in MSC viability, apoptosis, and exosome properties under diabetic conditions.
- To examine the expression of key genes and proteins involved in exosome biogenesis and release.
Main Methods:
- Human MSCs were cultured in diabetic or control sera for 7 days.
- Cell viability and apoptosis were assessed using MTT and flow cytometry.
- Gene expression (CD63, Alix, Rab27a, Rab27b, Rab8b) was quantified by real-time PCR.
- Exosome characteristics (acetylcholinesterase activity, size, zeta potential) were measured.
- CD63 protein localization was determined by immunofluorescence and Western blotting.
- Cell ultrastructure was examined via electron microscopy.
Main Results:
- Diabetic sera led to a slight decrease in MSC survival and increased apoptosis (p < 0.05).
- Expression of exosome-related genes (CD63, Alix, Rab27a, Rab27b, Rab8b) and CD63 protein significantly increased (p < 0.05).
- Exosomes from diabetic MSCs showed enhanced acetylcholinesterase activity, increased size, and decreased zeta potential (p < 0.05).
- Ultrastructural analysis revealed more cytoplasmic lipid vacuoles in diabetic MSCs.
Conclusions:
- Diabetic sera induce cellular stress and apoptosis in human MSCs.
- Diabetic conditions promote the production and alter the characteristics of MSC-derived exosomes.
- These findings highlight potential mechanisms by which diabetes affects MSC function and intercellular communication via exosomes.
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