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Photoreceptor apoptosis is an important pathogenesis of retinal degeneration and a primary cause of vision loss with limited treatment methods. Mesenchymal stem/stromal cells-derived small extracellular vesicles (MSC-sEVs) have shown therapeutic value in various ocular disorders. Recent studies have revealed that hypoxic preconditioning can improve the effectiveness of MSC-sEVs in tissue regeneration. However, whether hypoxic preconditioned MSC-sEVs (Hyp-sEVs) exert superior effects on photoreceptor protection relative to normoxic conditioned MSC-sEVs (Nor-sEVs) remains unclear. Here, we reported that Hyp-sEVs further improved retinal structure, recovered retinal function, and suppressed photoreceptor apoptosis in N-methyl-N-nitrosourea (MNU)-induced mouse model compared with Nor-sEVs. Hyp-sEVs also exhibited enhanced anti-apoptotic roles in MNU-provoked 661 W cell injury in vitro. We then analyzed the protein profiles of Nor-sEVs and Hyp-sEVs by LC-MS/MS and found that growth-associated protein 43 (GAP43) was enriched in Hyp-sEVs. The knockdown of GAP43 abolished the retinal therapeutic effects of Hyp-sEVs. Mechanistically, hypoxic stimulation-induced hypoxia-inducible factor-1α (HIF-1α) activation was responsible for preventing tripartite motif-containing protein 25 (TRIM25)-mediated GAP43 ubiquitination and degradation, leading to the upregulation of GAP43 in Hyp-sEVs. Together, our findings uncover the efficacy and mechanism of Hyp-sEVs-based photoreceptor protection and highlight the potential of Hyp-sEVs as optimized therapeutics for retinal degeneration. Graphical abstract
Photoreceptor apoptosis is an important pathogenesis of retinal degeneration and a primary cause of vision loss with limited treatment methods. Mesenchymal stem/stromal cells-derived small extracellular vesicles (MSC-sEVs) have shown therapeutic value in various ocular disorders. Recent studies have revealed that hypoxic preconditioning can improve the effectiveness of MSC-sEVs in tissue regeneration. However, whether hypoxic preconditioned MSC-sEVs (Hyp-sEVs) exert superior effects on photoreceptor protection relative to normoxic conditioned MSC-sEVs (Nor-sEVs) remains unclear. Here, we reported that Hyp-sEVs further improved retinal structure, recovered retinal function, and suppressed photoreceptor apoptosis in N-methyl-N-nitrosourea (MNU)-induced mouse model compared with Nor-sEVs. Hyp-sEVs also exhibited enhanced anti-apoptotic roles in MNU-provoked 661 W cell injury in vitro. We then analyzed the protein profiles of Nor-sEVs and Hyp-sEVs by LC-MS/MS and found that growth-associated protein 43 (GAP43) was enriched in Hyp-sEVs. The knockdown of GAP43 abolished the retinal therapeutic effects of Hyp-sEVs. Mechanistically, hypoxic stimulation-induced hypoxia-inducible factor-1α (HIF-1α) activation was responsible for preventing tripartite motif-containing protein 25 (TRIM25)-mediated GAP43 ubiquitination and degradation, leading to the upregulation of GAP43 in Hyp-sEVs. Together, our findings uncover the efficacy and mechanism of Hyp-sEVs-based photoreceptor protection and highlight the potential of Hyp-sEVs as optimized therapeutics for retinal degeneration. Graphical abstract
Extracellular vesicles (EVs) are nanosized particles that are released from cells and reflect the characteristics of the mother cell. Recently, the EVs have been used in several types of studies across many different fields. In the field of EV research, multiple cell culture and EV isolation techniques have been highlighted in importance. Various strategies, including exclusive component culture media, three-dimensional (3D) cultures, and hypoxic conditions, have been proposed for the cell culture to control function of the EVs. Ultracentrifugation, ultrafiltration, precipitation, and tangential flow filtration (TFF) have been utilized for EV isolation. Although isolated EVs have their own functionalities, several researchers are trying to functionalize EVs by applying various engineering approaches. Gene editing, exogenous, endogenous, and hybridization methods are the four well-known types of EV functionalization strategies. EV engineered through these processes has been applied in the field of regenerative medicine, including kidney diseases, osteoarthritis, rheumatoid arthritis, nervous system-related diseases, and others. In this review, it was focused on engineering approaches for EV functionalization and their applications in regenerative medicine.Graphical Abstract
Background and Objectives: Tacrolimus is a macrolide lactone compound derived from the bacterium Streptomyces tsukubensis, widely known as an immunosuppressant. In basic research, the effects of tacrolimus on osteogenic differentiation have been tested using mesenchymal stem cells. In this study, tacrolimus’s effects on the cellular survival and osteogenic differentiation of stem cell spheroids were investigated. Materials and Methods: Concave microwells were used to form stem cell spheroids in the presence of tacrolimus at final concentrations of 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL. A microscope was used to test cellular vitality qualitatively, and an assay kit based on water-soluble tetrazolium salt was used to measure cellular viability quantitatively. Alkaline phosphatase activity and an anthraquinone dye test for measuring calcium deposits were used to assess osteogenic differentiation. To assess the expression of osteogenic differentiation, a quantitative polymerase chain reaction, Western blot, and RNA sequencing were performed. Results: Spheroids across all concentrations maintained a relatively uniform and spherical shape. Cell viability assay indicated that tacrolimus, up to a concentration of 100 μg/mL, did not significantly impair cell viability within spheroids cultured in osteogenic media. The increase in calcium deposition, particularly at lower concentrations of tacrolimus, points toward an enhancement in osteogenic differentiation. There was an increase in COL1A1 expression across all tacrolimus concentrations, as evidenced by the elevated mean and median values, which may indicate enhanced osteogenic activity. Conclusions: This study showed that tacrolimus does not significantly impact the viability of stem cell spheroids in osteogenic media, even at high concentrations. It also suggests that tacrolimus may enhance osteogenic differentiation, as indicated by increased calcium deposition and COL1A1 expression. These findings advance our understanding of tacrolimus’s potential roles in tissue repair, regeneration, and stem cell-based therapeutic applications.
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