BACKGROUND Heart transplantation is a crucial intervention for severe heart failure,yet the challenge of organ rejection is significant.Bone marrow mesenchymal stem cells(BMSCs)and their exosomes have demonstrated pot...BACKGROUND Heart transplantation is a crucial intervention for severe heart failure,yet the challenge of organ rejection is significant.Bone marrow mesenchymal stem cells(BMSCs)and their exosomes have demonstrated potential in modulating T cells,dendtitic cells(DCs),and cytokines to achieve immunomodulatory effects.DCs,as key antigen-presenting cells,play a critical role in shaping immune responses by influencing T-cell activation and cytokine production.Through this modulation,BMSCs and their exosomes enhance graft tolerance and prolonging survival.AIM To explore the immunomodulatory effects of exosomes derived from BMSCs overexpressing microRNA-540-3p(miR-540-3p)on cardiac allograft tolerance,focusing on how these exosomes modulating DCs and T cells activity through the CD74/nuclear factor-kappaB(NF-κB)pathway.METHODS Rat models were used to assess the impact of miR-540-3p-enhanced exosomes on immune tolerance in cardiac allografts.MiR-540-3p expression was manipulated in BMSCs,and derived exosomes were collected and administered to the rat models post-heart transplantation.The study monitored expression levels of major histocompatibility complex II,CD80,CD86,and CD274 in DCs,and quantified CD4^(+)and CD8^(+)T cells,T regulatory cells,and cytokine profiles.RESULTS Exosomes from miR-540-3p-overexpressing BMSCs lead to reduced expression of immune activation markers CD74 and NF-κB p65 in DCs and T cells.Rats treated with these exosomes showed decreased inflammation and improved cardiac function,indicated by lower levels of pro-inflammatory cytokines(interleukin-1β,interferon-γ)and higher levels of anti-inflammatory cytokines(interleukin-10,transforming growth factorβ1).Additionally,miR-540-3p skewed the profiles of DCs and T cells towards immune tolerance,increasing the ratio of T regulatory cells and shifting cytokine secretion to favor graft acceptance.CONCLUSION Exosomes derived from BMSCs overexpressing miR-540-3p significantly enhance immune tolerance and prolong cardiac allograft survival by modulating the CD74/NF-κB pathway,which regulates activities of DCs and T cells.These findings highlight a promising therapeutic strategy to improve heart transplantation outcomes and potentially reduce the need for prolonged immunosuppression.展开更多
AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cry...AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.展开更多
基金Supported by the National Natural Science Foundation of China,No.82060299Medical Discipline Leader Project of Yunnan Provincial Health Commission,No.D-2019020+5 种基金Yunnan Provincial Government Ten Thousand Person-Top Young Talents Project,No.KH-SWRQNBJ-2019-002Clinical Medical Center of the First People’s Hospital of Yunnan Province,No.2021LCZXXF-XZ04 and No.2022LCZXKF-HX05Kunming Medical Joint Special Project-Outstanding Youth Cultivation Project,No.202101AY070001-034Kunming Medical Joint Special Project,No.202101AY070001-272Famous Doctor Project of“Xingdian Talent Support Plan”of Yunnan Province,No.XDYC-MY-2022-0037Yunnan Province 2023 Undergraduate Education and Teaching Reform Research Project,No.2023BKXJJG-F04002.
文摘BACKGROUND Heart transplantation is a crucial intervention for severe heart failure,yet the challenge of organ rejection is significant.Bone marrow mesenchymal stem cells(BMSCs)and their exosomes have demonstrated potential in modulating T cells,dendtitic cells(DCs),and cytokines to achieve immunomodulatory effects.DCs,as key antigen-presenting cells,play a critical role in shaping immune responses by influencing T-cell activation and cytokine production.Through this modulation,BMSCs and their exosomes enhance graft tolerance and prolonging survival.AIM To explore the immunomodulatory effects of exosomes derived from BMSCs overexpressing microRNA-540-3p(miR-540-3p)on cardiac allograft tolerance,focusing on how these exosomes modulating DCs and T cells activity through the CD74/nuclear factor-kappaB(NF-κB)pathway.METHODS Rat models were used to assess the impact of miR-540-3p-enhanced exosomes on immune tolerance in cardiac allografts.MiR-540-3p expression was manipulated in BMSCs,and derived exosomes were collected and administered to the rat models post-heart transplantation.The study monitored expression levels of major histocompatibility complex II,CD80,CD86,and CD274 in DCs,and quantified CD4^(+)and CD8^(+)T cells,T regulatory cells,and cytokine profiles.RESULTS Exosomes from miR-540-3p-overexpressing BMSCs lead to reduced expression of immune activation markers CD74 and NF-κB p65 in DCs and T cells.Rats treated with these exosomes showed decreased inflammation and improved cardiac function,indicated by lower levels of pro-inflammatory cytokines(interleukin-1β,interferon-γ)and higher levels of anti-inflammatory cytokines(interleukin-10,transforming growth factorβ1).Additionally,miR-540-3p skewed the profiles of DCs and T cells towards immune tolerance,increasing the ratio of T regulatory cells and shifting cytokine secretion to favor graft acceptance.CONCLUSION Exosomes derived from BMSCs overexpressing miR-540-3p significantly enhance immune tolerance and prolong cardiac allograft survival by modulating the CD74/NF-κB pathway,which regulates activities of DCs and T cells.These findings highlight a promising therapeutic strategy to improve heart transplantation outcomes and potentially reduce the need for prolonged immunosuppression.
基金Supported by Grants from VINNMER,Lundin foundation, R&D Funds from Stockholm County and Karolinska Institutet (ALF),and the Swedish Research Council
文摘AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.
