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Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

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World Journal of stem cells 2020年第10期12卷 1171-1183页

作者： Kristine Karla Freude Sarkis Saruhanian Alanna McCauley Colton Paterson Madeleine Odette Annika Oostenink Poul Hyttel Mark Gillies Henriette Haukedal Miriam Kolko Department of Veterinary and Animal Sciences Group of Stem Cell Models for Studies of Neurodegenerative DiseasesSection for Pathobiological SciencesFaculty of Health and Medical SciencesUniversity of CopenhagenFrederiksberg 1870Denmark Department of Drug Design and Pharmacology University of CopenhagenCopenhagen 2100Denmark Save Sight Institute South BlockSydney Eye HospitalSydney 2000Australia Department of Ophthalmology Copenhagen University HospitalRigshospitalet-GlostrupGlostrup 2600Denmark

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from *** permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller *** To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream *** In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D *** progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was *** Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal *** retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as *** Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

关键词： human induced pluripotent stem cells Retinal organoids Retinal ganglion cells Müller glia Progenitors Cell-type enrichment

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific human induced pluripotent stem cells

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Chinese Medical Sciences Journal 2017年第3期32卷 135-144页

作者：何琼王惠荟程涛袁卫平马钰波蒋永平任志华中国医学科学院北京协和医学院方舟生物医药研发中心苏州215126 苏州方舟基因药业有限公司苏州215126 中国医学科学院北京协和医学院干细胞医学中心血液病医院实验血液学国家重点实验室天津300020 长岛高科技孵化器iCell基因治疗有限责任公司研究开发部石溪大学医学院病理部澳门科技大学澳门药物及健康应用研究院

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA *** The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

关键词： hemophilia B human induced pluripotent stem cells CRISPR/Cas9 genetic correction hepatic differentiation

Three-dimensional hydrogel culture conditions promote human induced pluripotent stem cells differentiated hepatocytes

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Three-dimensional hydrogel culture conditions promote human ...

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

第二十四次全国中西医结合肝病学术会议

作者： Ying Luo Yingtang Gao Cheng Lou Sui Zhang Zhengyan Zhu Qianzhe Xing Peng Wang Tong Liu Chenglong Li Li Jing Hui Liu Wenxia Shi Zhi Du Key Laboratory of Artificial Cell Institute of Hepatobiliary DiseaseThe Third Central Hospital of Tianjin China Department of Hepatobiliary Surgery The Third Central Hospital of Tianjin Department of Cardiology The University of Texas MD Anderson Cancer Center

Background & Aims:The use of differentiated hepatocytes from hiPSCs is increasingly favored for BAL,but,inferior function limited it *** this study,we aimed to establish a 3D condition to mimic the environment that of hepatogenesis in vivoforlarge-scale culture and higher throughput ***:We utilize a synthetic hydrogel to create the HLCs-based spheroids in the 3D condition and evaluate the maturity of spheroids ***:We differentiated hiPSCs to DE cells with EB formation in monolayer condition in the first stage,and then dissociatedhiPSCs-DE into single cells to implante into hydrogel via self-aggregatingto form *** HLCs-based spheroids showed expression of hepatocyte *** ALB secretion,Urea production and glycogen synthesis by HLCs in 3D conditions are significantly higher than the cells in 2D conditions,or *** addition,metabolic activities of the CYP450 enzymes were also improved while cells differented in 3D ***:This 3Dhydrogel conditions can promote human induced pluripotent stem cellsdifferentiated hepatocytes,and it may be have the potential application of fordifferentiated hepatocytes from hiPSCs as a source for BAL.

关键词： human induced pluripotent stem cells differentiation 3D condition hydrogel

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World Journal of stem cells 2024年第5期16卷 512-524页

作者： Hang Zhang Ling-Zi Wu Zhen-Yu Liu Zi-Bing Jin Beijing Institute of Ophthalmology Beijing Tongren Eye CenterBeijing Tongren HospitalCapital Medical UniversityBeijing 100730China

BACKGROUND human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease ***,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent *** To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation *** We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK ***-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC *** and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation ***,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and *** The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic ***,hiPSC-derived EVs were involved in various biological processes,including cell junction and *** HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation ***,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.

关键词： Cell junction Cellular differentiation Extracellular vesicle human induced pluripotent stem cells Transcriptomics Proteomics

Rebuilding insight into the pathophysiology of Alzheimer’s disease through new blood-brain barrier models

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Neural Regeneration Research 2024年第9期19卷 1954-1960页

作者： Kinya Matsuo Hideaki Nshihara Yamaguchi University Graduate School of Medicine YamaguchiJapan Department of Neurotherapeutics Yamaguchi UniversityUbeJapan

The blood-brain barrier is a unique function of the microvasculature in the brain parenchyma that maintains homeostasis in the central nervous ***-brain barrier breakdown is a common pathology in various neurological diseases,such as Alzheimer’s disease,stroke,multiple sclerosis,and Parkinson’s ***,it has been considered a consequence of neuroinflammation or neurodegeneration,but recent advanced imaging techniques and detailed studies in animal models show that blood-brain barrier breakdown occurs early in the disease process and may precede neuronal ***,the blood-brain barrier is attractive as a potential therapeutic target for neurological diseases that lack effective *** elucidate the molecular mechanism underlying blood-brain barrier breakdown and translate them into therapeutic strategies for neurological diseases,there is a growing demand for experimental models of human origin that allow for functional ***,several human induced pluripotent stem cell-derived blood-brain barrier models have been established and various in vitro blood-brain barrier models using microdevices have been *** in the Alzheimer’s disease field,the human evidence for blood-brain barrier dysfunction has been demonstrated and human induced pluripotent stem cell-derived blood-brain barrier models have suggested the putative molecular mechanisms of pathological blood-brain *** this review,we summarize recent evidence of blood-brain barrier dysfunction in Alzheimer’s disease from pathological analyses,imaging studies,animal models,and stem cell ***,we discuss the potential future directions for blood-brain barrier research.

关键词： Alzheimer’s disease blood-brain barrier human induced pluripotent stem cells

Role of induced pluripotent stem cells in diagnostic cardiology

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World Journal of stem cells 2021年第5期13卷 331-341页

作者： Steven B Karch Vittorio Fineschi Pietro Francia Matteo Scopetti Martina Padovano Federico Manetti Alessandro Santurro Paola Frati Massimo Volpe School of Medicine University of NevadaLas VegasNV 89102United States Department of Anatomical HistologicalForensic and Orthopaedic SciencesSapienza University of RomeRome 00185Italy Division of Cardiology Department of Clinical and Molecular MedicineSapienza University of RomeSt.Andrea HospitalVia di Grottarossa103500189 RomeItaly Department SAIMLAL Sapienza University of RomaRome 00185Italy Department of Clinical and Molecular Medicine Sapienza University of RomeRome 00197Italy

Ethical concerns about stem cell-based research have delayed important advances in many areas of medicine,including *** introduction of induced pluripotent stem cells(iPSCs)has supplanted the need to use human stem cells for most purposes,thus eliminating all ethical *** then,many new avenues have been opened in cardiology research,not only in approaches to tissue replacement but also in the design and testing of antiarrhythmic *** methodology has advanced to the point where induced human cardiomyocyte cell lines can now also be obtained from commercial sources or tissue *** studies with readily available iPSCs have generally confirmed that their behavioral characteristics accurately predict the behavior of beating cardiomyocytes in *** a result,iPSCs can provide new ways to study arrhythmias and heart disease in general,accelerating the development of new,more effective antiarrhythmic drugs,clinical diagnoses,and personalized medical *** focus on producing cardiomyocytes that can be used to replace damaged heart tissue has somewhat diverted interest in a host of other *** manuscript is intended to provide non-specialists with a brief introduction and overview of the research carried out in the field of heart rhythm disorders.

关键词： human induced pluripotent stem cells Diagnostic cardiology Heart rhythm disorders Microelectrode array stem cell research Ethical principles

human pluripotent stem cells:Towards therapeutic development for the treatment of lifestyle diseases

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World Journal of stem cells 2016年第2期8卷 56-61页

作者： Miwako Nishio Masako Nakahara Akira Yuo Kumiko Saeki Research Institute National Center for Global Health and Medicine PRESTO Japan Science and Technology Agency

There are two types of human pluripotent stem cells: Embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs),both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iP *** is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living *** lifestyle diseases,we have already several therapeutic options,and thus,development of human pluripotent stem cell-based therapeutics tends to be ***,human pluripotent stem cells can contribute to the development of new therapeutics in this *** we will show,there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are *** those cases,immunologically rejections of ESC-or allogenic iP SC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic *** utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise *** example,clinical specimens of human classical brown adipocytes(BAs),which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders,are unobtainable from living individuals due to scarcity,fragility and ethical ***,BA can easily be produced from human pluripotent stem *** this review,we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases.

关键词： Arteriostenosis human embryonic stem cells Glucose intolerance human induced pluripotent stem cells Brown adipose tissue

Skeletal muscle generated from induced pluripotent stem cells-induction and application

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World Journal of stem cells 2017年第6期9卷 89-97页

作者： Yuko Miyagoe-Suzuki Shin’ichi Takeda Department of Molecular Therapy National Institute of NeuroscienceNational Center of Neurology and PsychiatryKodairaTokyo 187-8502Japan

human induced pluripotent stem cells(hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patientiPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle diseaseiPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage:(1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease;(2) setting up a highthroughput drug screening system; and(3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.

关键词： human induced pluripotent stem cells Skeletal muscle Transplantation Disease Modeling Muscle progenitors Muscular dystrophy MYOD

A novel xeno-free and feeder-cell-free system for human pluripotent stem cell culture

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Protein & Cell 2012年第1期3卷 51-59页

作者： Qihui Wang Xiaoning Mou Henghua Cao Qingzhang Meng Yanni Ma Pengcheng Han Junjie Jiang Hao Zhang Yue Ma National Laboratory of Biomacromolecules Institute of BiophysicsChinese Academy of SciencesBeijing 100101China Graduate School of the Chinese Academy of Sciences Beijing 100080China Department of Surgery Cardiovascular InstituteFu Wai Heart HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100037China

While human induced pluripotent stem cells(hiPSCs)have promising applications in regenerative medicine,most of the hiPSC lines available today are not suitable for clinical applications due to contamination with nonhuman materials,such as sialic acid,and potential pathogens from animal-product-containing cell culture *** several xeno-free cell culture systems have been established recently,their use of human fibroblasts as feeders reduces the clinical potential of hiPSCs due to batch-to-batch variation in the feeders and time-consuming preparation *** this study,we have developed a xeno-free and feeder-cell-free human embryonic stem cell(hESC)/hiPSC culture system using human plasma and human placenta *** system maintains the self-renewing capacity and pluripotency of hESCs for more than 40 *** iPSCs were also derived from human dermal fibroblasts using this culture system by overexpressing three transcription factors—Oct4,Sox2 and *** culture system developed here is inexpensive and suitable for large scale production.

关键词： human embryonic stem cells human induced pluripotent stem cells reprogramming xeno-free and feeder-cell-free culture system