Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional(3D)architecture of tissues and deciphering cellular spatial interactions under physiologic...Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional(3D)architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions.Deciphering the spatial interaction of leptin receptor-expressing(LepR^(+))stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue.In this study,we introduce an optimized protocol for the 3D analysis of skeletal tissues,enabling the visualization of hematopoietic and stromal cells,especially LepR+stromal cells,within optically cleared bone hemisections.Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae.The protocol entails tissue fixation,decalcification,and cryosectioning to reveal the marrow cavity.Completed within approximately 12 days,this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals.The bone hemisections are compatible with diverse antibody labeling strategies.Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software,assessing a spectrum of parameters.With proper storage,the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months.This protocol is robust,straightforward to implement,and highly reproducible,offering a valuable tool for tissue architecture and cellular interaction studies.展开更多
The ambiguity of etiology makes temporomandibular joint osteoarthritis(TMJOA)“difficult-to-treat”.Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management.Nonetheless,challenges...The ambiguity of etiology makes temporomandibular joint osteoarthritis(TMJOA)“difficult-to-treat”.Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management.Nonetheless,challenges such as low yields and insignificant efficacy of current exosome therapies necessitate significant advances.Addressing lower strontium(Sr)levels in arthritic synovial microenvironment,we studied the effect of Sr element on exosomes and miRNA selectively loading in synovial mesenchymal stem cells(SMSCs).Here,we developed an optimized system that boosts the yield of SMSC-derived exosomes(SMSCEXOs)and improves their miRNA profiles with an elevated proportion of beneficial miRNAs,while reducing harmful ones by pretreating SMSCs with Sr.Compared to untreated SMSC-EXOs,Sr-pretreated SMSC-derived exosomes(Sr-SMSC-EXOs)demonstrated superior therapeutic efficacy by mitigating chondrocyte ferroptosis and reducing osteoclast-mediated joint pain in TMJOA.Our results illustrate Alix’s crucial role in Sr-triggered miRNA loading,identifying miR-143-3p as a key anti-TMJOA exosomal component.Interestingly,this system is specifically oriented towards synovium-derived stem cells.The insight into trace elementdriven,site-specific miRNA selectively loading in SMSC-EXOs proposes a promising therapeutic enhancement strategy for TMJOA.展开更多
基金National Natural Science Foundation of China(grant number 82272563 to B.S.)National Science and Technology Major Project of the Ministry of Science and Technology of China(grant number 2023ZD0501202 to B.S.)+4 种基金institutional grants allocated to the National Institute of Biological Sciences,Beijing(NIBS)from the Chinese Ministry of Science and Technology,Beijing Municipal Commission of Science and Technology,and Tsinghua Universitythe support from China Pharmaceutical University(grant number 3150140001 to S.F.)National Natural Science Foundation of China(grant numbers 82203653 to S.F.,82371957 to L.W.,and 82371956 to X.C.)Beijing Municipal Public Welfare Development and Reform Pilot Project for Medical Research Institutes(grant number JYY2023-8 to X.C.)Research Grants Council of University Grants Committee Hong Kong(grant numbers 14113723,14108720,14121721,14202920,N_CUHK472/22,C7030-18G,T13-402/17-N,and AoE/M-402/20)。
文摘Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional(3D)architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions.Deciphering the spatial interaction of leptin receptor-expressing(LepR^(+))stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue.In this study,we introduce an optimized protocol for the 3D analysis of skeletal tissues,enabling the visualization of hematopoietic and stromal cells,especially LepR+stromal cells,within optically cleared bone hemisections.Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae.The protocol entails tissue fixation,decalcification,and cryosectioning to reveal the marrow cavity.Completed within approximately 12 days,this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals.The bone hemisections are compatible with diverse antibody labeling strategies.Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software,assessing a spectrum of parameters.With proper storage,the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months.This protocol is robust,straightforward to implement,and highly reproducible,offering a valuable tool for tissue architecture and cellular interaction studies.
基金supported by National Natural Science Foundation of China(Nos.82271019,82472149,82471024)Sichuan Science and Technology Program(No.24ZDYF0099)Research and Develop Program,West China Hospital of Stomatology Sichuan University(RD-03-202101)to J.W.
文摘The ambiguity of etiology makes temporomandibular joint osteoarthritis(TMJOA)“difficult-to-treat”.Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management.Nonetheless,challenges such as low yields and insignificant efficacy of current exosome therapies necessitate significant advances.Addressing lower strontium(Sr)levels in arthritic synovial microenvironment,we studied the effect of Sr element on exosomes and miRNA selectively loading in synovial mesenchymal stem cells(SMSCs).Here,we developed an optimized system that boosts the yield of SMSC-derived exosomes(SMSCEXOs)and improves their miRNA profiles with an elevated proportion of beneficial miRNAs,while reducing harmful ones by pretreating SMSCs with Sr.Compared to untreated SMSC-EXOs,Sr-pretreated SMSC-derived exosomes(Sr-SMSC-EXOs)demonstrated superior therapeutic efficacy by mitigating chondrocyte ferroptosis and reducing osteoclast-mediated joint pain in TMJOA.Our results illustrate Alix’s crucial role in Sr-triggered miRNA loading,identifying miR-143-3p as a key anti-TMJOA exosomal component.Interestingly,this system is specifically oriented towards synovium-derived stem cells.The insight into trace elementdriven,site-specific miRNA selectively loading in SMSC-EXOs proposes a promising therapeutic enhancement strategy for TMJOA.
