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Long-term in vivo chimeric cells tracking in non-human primate

Protein & Cell 2024年第3期15卷 207-222页

作者： Junmo Wu Yu Kang Xiang Luo Shaoxing Dai Yuxi Shi Zhuoyao Li Zengli Tang Zhenzhen Chen Ran Zhu Pengpeng Yang Zifan Li Hong Wang Xinglong Chen Ziyi Zhao Weizhi ji Yuyu Niu State Key Laboratory of Primate Biomedical Research Institute of Primate Translational MedicineKunming University of Science and TechnologyKunming 650500China Yunnan Key Laboratory of Primate Biomedical Research Kunming 650500China Faculty of Life Science and Technology Kunming University of Science and TechnologyKunming 650500China

Non-human primates(NHPs)are increasingly used in preclinical trials to test the safety and efficacy of biotech-nology therapies.Nonetheless,given the ethical issues and costs associated with this model,it would be highly advantageous to use NHP cellular models in clinical studies.However,developing and maintaining the naive state of primate pluripotent stem cells(PSCs)remains difficult as does in vivo detection of PSCs,thus limiting biotech-nology application in the cynomolgus monkey.Here,we report a chemically defined,xeno-free culture system for culturing and deriving monkey PSCs in vitro.The cells display global gene expression and genome-wide hypometh-ylation patterns distinct from monkey-primed cells.We also found expression of signaling pathways components that may increase the potential for chimera formation.Crucially for biomedical applications,we were also able to integrate bioluminescent reporter genes into monkey PsCs and track them in chimeric embryos in vivo and in vitro.The engineered cells retained embryonic and extra-embryonic developmental potential.Meanwhile,we generated a chimeric monkey carrying bioluminescent cells,which were able to track chimeric cells for more than 2 years in living animals.Our study could have broad utility in primate stem cell engineering and in utilizing chimeric monkey models forclinical studies.

关键词： pluripotent stem cells bioluminescence imaging non-human primates

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Quantitative Evaluation of bioluminescence imaging Based on Radiative Transfer Equation

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Chinese Journal of Biomedical Engineering(English Edition) 2016年第1期25卷 39-46页

作者： KAN Xing-xing CHEN Chun-xiao GONG Rong-fang Department of Biomedical Engineering Nanjing University of Aeronautics & Astronautics Department of Mathematics Nanjing University of Aeronautics & Astronautics

bioluminescence imaging is a kind of emerging detection technology at cellular,molecular and genetic level.The most popular bioluminescence imaging model is diffusion approximation(DA).However,because of the ill-posedness of the DA-based inverse problem and the instability of reconstruction algorithms,the location accuracy of the reconstructed sources is low.Radiative transfer equation(RTE),which considers the direction of the photon migration and the effect of absorption and scattering in tissues,can accurately express the transmission of bioluminescent photons through the tissues.In this paper,we studied the bioluminescence imaging based on the RTE.2D simulations were performed,and quantitative evaluation was given by the absolute source position error,the relative source area error and the minimum bounding box.The results of the experiment showed that the imaging quality based on RTE was better than that one based on DA.

关键词： bioluminescence imaging radiative transfer equation discontinuous Galerkin method forward problem inverse problem

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AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo

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Genes & Diseases 2022年第4期9卷 1074-1085页

作者： Chi Heon Kim Colleen Oliver Hamid Dar Hicham Drissi Steven M.Presciutti Department of Orthopaedics Emory University School of MedicineAtlantaGA 30329USA Atlanta Veteran Affairs Medical Center DecaturGA 30030USA

Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration,is a fascinating and promising tool to manipulate the complex physiology of the disc.Viral vectors based on the adeno-associated virus(AAV)have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism,transduction efficiency,and relative toxicity have not yet been performed in the disc in vivo.Herein,we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes,injection volumes,titers,promoters,and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus(NP)cells in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides.Finally,to our knowledge,we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc,which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery.Taken together,these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

关键词： Adeno-associated virus bioluminescence imaging Cell permeabilization peptide Intervertebral disc Lightsheet microscopy Nucleus pulposus transduction Tissue clearing

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Optimizing a high-sensitivity NanoLuc-based bioluminescence system for in vivo evaluation of antimicrobial treatment

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mLife 2023年第4期2卷 462-478页

作者： Weilong Shang Zhen Hu Mengyang Li Yuting Wang Yifan Rao Li Tan Juan Chen Xiaonan Huang Lu Liu He Liu Zuwen Guo Huagang Peng Yi Yang Qiwen Hu Shu Li Xiaomei Hu Jiao Zou Xiancai Rao Department of Microbiology College of Basic Medical SciencesKey Laboratory of Microbial Engineering Under the Educational Committee in ChongqingArmy Medical University(Third Military Medical University)ChongqingChina Department of Microbiology School of MedicineChongqing UniversityChongqingChina Department of Emergency Medicine Xinqiao HospitalArmy Medical University(Third Military Medical University)ChongqingChina Department of Pharmacy Xinqiao HospitalArmy Medical University(Third Military Medical University)ChongqingChina Department of Military Cognitive Psychology School of PsychologyArmy Medical University(Third Military Medical University)ChongqingChina.

Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects.However,high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches.Here,we report that NanoLuc(Nluc)showed better performance than LuxCDABE in bacteria.However,the retention rate of plasmid constructs in bacteria was low.To construct stable Staphylococcus aureus reporter strains,a partner protein enolase(Eno)was identified by screening of S.aureus strain USA300 for fusion expression of Nluc-based luciferases,including Nluc,Teluc,and Antares2.Different substrates,such as hydrofurimazine(HFZ),furimazine(FUR),and diphenylterazine(DTZ),were used to optimize a stable reporter strain/substrate pair for BL imaging.S.aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S.aureus in vivo,with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys.USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics.The optimized S.aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.

关键词： antimicrobial evaluation bioluminescence imaging Nluc-based luciferases Staphylococcus aureus therapeutic efficacy

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The 9L^(LUC)/Wistar rat glioma model is not suitable for immunotherapy

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Neural Regeneration Research 2012年第18期7卷 1406-1411页

作者： Liping Yang Jingxiang Zhao Guihong Zhou Yunfang Wang Lusi Li Hongfeng Yuan Xue Nan Lidong Guan Xuetao Pei Department of Medicine the 263 Hospital Beijing 101149 China Department of Neurology South West Hospital the Third Military Medical University of Chinese PLA Chongqing 400038 China Department of Stem Cell and Regenerative Medicine Beijing Institute of Transfusion Medicine Beijing 100850 China Department of Neurosurgery the 263 Hospital Beijing 101149 China

The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9L^LUC glioma cells syngenic to Fischer 344 （F344） rats. bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9L^LUC/F344 rats, and tumor regression was found in some 9L^LUC/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9L^LUC/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9L^LUC/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.

关键词： 9L cells glioma F344 rats Wistar rats animal model bioluminescence imaging immune response neural regeneration

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A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

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Plant Communications 2022年第1期3卷 6-20页

作者： Ayumi Matsumoto Titus Schluter Katharina Melkonian Atsushi Takeda Hirofumi Nakagami Akira Mine Research Organization of Science and Technology Ritsumeikan UniversityShiga 525-8577Japan Basic Immune System of Plants Max Planck Institute for Plant Breeding ResearchCologne 50829Germany College of Life Sciences Ritsumeikan UniversityShiga 525-8577Japan JST PRESTO Kawaguchi-shiSaitama 332-0012Japan Laboratory of Plant Pathology Graduate School of AgricultureKyoto UniversityKyoto 606-8502Japan

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays.Here,we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants.We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABEluciferase operon into a specific genomic location found ubiquitously across bacterial phyla.These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas,Rhizobium(Agrobacterium),and Ralstonia.Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv.tomato DC3000(Pto-lux)reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana,Solanum lycopersicum,Nicotiana benthamiana,and Marchantia polymorpha.We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains.Importantly,quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth,with a dynamic range of four orders of magnitude.Moreover,macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues.In conclusion,our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions.

关键词： plant immunity bacterial virulence bioluminescence imaging Pseudomonas syringae Arabidopsis thaliana Marchantia polymorpha

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Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation

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Chinese Chemical Letters 2016年第4期27卷 550-554页

作者： Ming-Liang Yuan Tian-Yu Jiang Lu-Pei Du Min-Yong Li Department of Medicinal Chemistry Key Laboratory of Chemical Biology (MOE) School of Pharmacy Shandong University

Replacement of the methylene group at the C-8 position with an extended electronic conjugation is a new promising method to develop red-shifted coelenterazine derivatives. In this paper, we have described an oxygen-containing coelenterazine derivative with a significant red-shifted（63 nm）bioluminescence signal maximum relative to coelenterazine 400a（Deep Blue C^（TM）, 1）. In cell imaging, the sulfur-containing coelenterazine derivative displayed a significantly（1.77 ± 0.09;P≤0.01） higher luminescence signal compared to coelenterazine 400 a and the oxygen-containing coelenterazine derivative exhibited a slightly（0.74 ± 0.08; P≤0.05） lower luminescence signal. It is beneficial to understand further the underlying mechanisms of bioluminescence.

关键词： bioluminescence imaging Coelenterazine derivatives Relative quantum yield Chemiluminescence Red-shifted

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Real-time monitoring of catheter-related biofilm infection in mice

Real-time monitoring of catheter-related biofilm infection i...

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第十二届全国化疗药理学术研讨会

作者： Xu Liu Yun Cai Department of Clinical Pharmacology PLA General Hospital Medicine Clinical Research Center PLA General Hospital

Objective:This study was to establish a mice model for catheter-related biofilm infection suitable to bioluminescence imaging（BLI）.Methods:Biofilms of Pseudomonas aeruginosa（P aeruginosa）Xen5 to be grown on catheter disks in vitro and implanted into mice to induce catheter-related biofilm infections in vivo.Biofilm formation in vitro and in vivo was real-time monitored during the study period using BLI.The images were analysed,and the relationship between integrated brightness（IB）and viable bacterial count（VBC）was compared.In addition,we monitored the change in white blood cells（WBCs）during the biofilm infection.Results:The levels of IB and VBC in biofilm disks in vitro were highest at 24 h after P.aeruginosa Xen5 inoculation,IB of biofilm in implanted mice model was increased until 24 h.A good relationship was observed between IB and VBC in vitro by linear regression analysis.The actual number of VBC in the infected catheter disks at day 7 of implantation was very close to the estimated number according to the formula of linear regression from in vitro study.The number of WBCs in the control group was gradually increased from day 1 to day 5 but restored to normal level after that,while their number in the infection group was gradually increased during the study period.The number of WBCs on day 7 was significantly higher in the infection group than control group.Conclusion:bioluminescence imaging is a simple,fast and sensitive method to measure the catheter biofilm infection in mice.

关键词： bioluminescence imaging mice catheter-related infection biofilm

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Mature dendritic cell derived from cryopreserved immature dendritic cell shows impaired homing ability and reduced antiviral therapeutic effects

Mature dendritic cell derived from cryopreserved immature de...

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第十一届全国免疫学学术大会

作者： Zhan Linsheng Zhou Qianqian Zhang Yulong Wang Xiaohui Beijing transfusion institute

Objective: Cryopreservation is critical in reducing redundant operations and also in quality control in dendritic cell(DC) therapy. Full maturation and efficient homing of DCs to T cell-region constitute a crucial aspect of DC immunotherapy;however, the in vivo migration and distribution pattern, as well as the anti-viral effect of DC matured from cryopreserved immature DCs(cryoim-m DCs) remain to be revealed.Methods: In the present study, we compared cryoim-m DCs with DCs matured from fresh immature DCs(fm DCs) in the aspects of phenotypes, in vivo homing capacities as well as the anti-viral therapeutic effects to further clarify the effect of cryopreservation on DC-based cytotherapy. Results: The results show that cryopreservation impair the homing ability of DCs which was associated with the reduced expression of CCR7 and disturbed cytoskeleton arrangement. Moreover, the antigen-specific CD8 T cell response induced by cryoim-m DCs was much weaker than that induced by fm DCs in both the spleen and liver draining lymph nodes, which provided reduced protection from viral invasions. Conclusions: In conclusion, cryopreservation is a good method to keep the viability of immature DCs, however, the in vivo homing capacity and anti-viral therapeutic effect of DC matured from frozen immature DCs were hindered to some extent.

关键词： DCs cryopreservation migration bioluminescence imaging anti-viral effects

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Co-loading of PolyMet and Doxorubicin via Core-shell Structure Liposomes for Treating DOX-resistant Breast cancer

Co-loading of PolyMet and Doxorubicin via Core-shell Structu...

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2018年第十二届中国药物制剂大会

作者：俞建东 Chao-yuan TANG Shuang ZHENG Luhui FAN Yang XIONG College of Pharmaceutical Science Zhejiang Chinese Medical University

Objectives:Polymeric Metformin（PolyMet） has been reported to be a potential anticancer drug and carrier which has the synergistic effect with some chemotherapeutics or siRNA to treat cancer. It was reported that guanidinium-rich molecular transporters could penetrate cells and overcome efflux-mediated multidrug resistance（MDR） so whether PolyMet, which with rich guanidinium, has the effect for overcoming MDR? In this study, according to the theoretical support that intracellular pharmacokinetic（PK） of D-luciferin potassium salt（a kind of ABC substrate） related to the activity of ABC transporter "drug pump", the PK parameters of D-luciferin potassium salt in MCF-7/DOXFluc cells treated with PolyMet or not was used to evaluate whether PolyMet could overcome efflux-mediated MDR in vitro, and the MDR reversing effect in vivo was further confirmed by checking its anti-tumor efficiency in MCF-7/DOX bearing BALB/c Nude mice when combined with DOX. In addition, in order to further improve the antitumor efficiency and lower the toxicity of DOX, PolyMet was also used as carrier to co-load DOX into the core-shell structure liposomes. Methods:MCF-7/DOXFluc cell line with stable over-expressed luciferase was established. bioluminescence imaging（BLI） was used to real-time monitor the efflux kinetics of D-luciferin potassium salt（ABC substrate） before and after MCF-7/DOXFluc cells treated with PolyMet. For co-load PolyMet and DOX in liposomes, DOX was first conjugated to polyglutamic acid（PGA） to form anionic PGA-DOX which was electrostatically complexed with the cationic PolyMet. The nano-sized core was then coated with cationic liposomes composed of（2, 3-Dioleoyloxy-propyl）-trimethylammonium（DOTAP）/Cholesterol/DSPE-PEG to form the core-shell structure liposomes（LPPD）. Finally, in order to verify whether PolyMet could improve the anti-cancer efficacy, MCF-7/DOX bearing BALB/c Nude mice were given different doses of free doxorubicin（1 mg/kg or 5 mg/kg） or LPPD（1 mg/kg）. Results:The results of BLI showed that the efflux of D-luc

关键词： Multi-Drug Resistance Doxorubicin PolyMet Liposome bioluminescence imaging

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