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Single-cell omics: experimental workflow, data analyses and applications

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Science China Life sciences 2025年第1期68卷 5-102页

作者： Fengying Sun Haoyan Li Dongqing Sun Shaliu Fu Lei Gu Xin Shao Qinqin Wang Xin Dong Bin Duan Feiyang Xing Jun Wu Minmin Xiao Fangqing Zhao Jing-Dong J.Han Qi Liu Xiaohui Fan Chen Li Chenfei Wang Tieliu Shi department of Clinical Laboratory the Affiliated Wuhu Hospital of East China Normal University (The Second People's Hospital of Wuhu City) Pharmaceutical Informatics Institute College of Pharmaceutical SciencesZhejiang University Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration (Tongji University) Ministry of EducationOrthopaedic DepartmentTongji HospitalBioinformatics DepartmentSchool of Life Sciences and TechnologyTongji University Frontier Science Center for Stem Cells School of Life Sciences and TechnologyTongji University Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine Shanghai East HospitalBioinformatics DepartmentSchool of Life Sciences and TechnologyTongji University Research Institute of Intelligent Computing Zhejiang Lab Shanghai Research Institute for Intelligent Autonomous Systems Center for Single-cell Omics School of Public HealthShanghai Jiao Tong University School of Medicine National Key Laboratory of Chinese Medicine Modernization Innovation Center of Yangtze River DeltaZhejiang University Center for Bioinformatics and Computational biology Shanghai Key Laboratory of Regulatory Biologythe Institute of Biomedical Sciences and School of Life SciencesEast China Normal University Beijing Institutes of Life Science Chinese Academy of Sciences Peking-Tsinghua Center for Life sciences Academy for Advanced Interdisciplinary StudiesCenter for Quantitative Biology (CQB)Peking University Zhejiang Key Laboratory of Precision Diagnosis and Therapy for Major Gynecological Diseases Women's HospitalZhejiang University School of Medicine Key Laboratory of Advanced Theory and Application in Statistics and Data Science-MOE School of StatisticsEast China Normal University

Cells are the fundamental units of biological systems and exhibit unique development trajectories and molecular features. Our exploration of how the genomes orchestrate the formation and maintenance of each cell, and control the cellular phenotypes of various organismsis, is both captivating and intricate. Since the inception of the first single-cell RNA technology, technologies related to single-cell sequencing have experienced rapid advancements in recent years. These technologies have expanded horizontally to include single-cell genome, epigenome,proteome, and metabolome, while vertically, they have progressed to integrate multiple omics data and incorporate additional information such as spatial sc RNA-seq and CRISPR screening. Single-cell omics represent a groundbreaking advancement in the biomedical field,offering profound insights into the understanding of complex diseases, including cancers. Here, we comprehensively summarize recent advances in single-cell omics technologies, with a specific focus on the methodology section. This overview aims to guide researchers in selecting appropriate methods for single-cell sequencing and related data analysis.

关键词： single-cell sequencing genome epigenome proteomics metabolomics multimodal spatial transcriptomics CRISPR screening

Spontaneous emergence of overgrown molar teeth in a colony of Prairie voles(Microtus ochrogaster)

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International Journal of Oral Science 2015年第1期7卷 23-26页

作者： Andrew H Jheon Michaela Prochazkova Michael Sherman Devanand S Manoli Nirao M Shah Lawrence Carbone Ophir Klein department of orofacial sciences and program in craniofacial and Mesenchymal biology University of California San Francisco (UCSF) department of Anthropology and Human Genetics Faculty of Science Charles University in Prague Laboratory Animal Resource Center UCSF department of Psychiatry UCSF department of Anatomy UCSF

Continuously growing incisors are common to all rodents, which include the Microtus genus of voles. However, unlike many rodents, voles also possess continuously growing molars. Here, we report spontaneous molar defects in a population of Prairie voles （Microtus ochrogaster）. We identified bilateral protuberances on the ventral surface of the mandible in several voles in our colony. In some cases, the protuberances broke through the cortical bone. The mandibular molars became exposed and infected, and the maxillary molars entered the cranial vault. Visualisation upon soft tissue removal and microcomputed tomography （microCT） analyses confirmed that the protuberances were caused by the overgrowth of the apical ends of the molar teeth. We speculate that the unrestricted growth of the molars was due to the misregulation of the molar dental stem cell niche. Further study of this molar phenotype may yield additional insight into stem cell regulation and the evolution and development of continuously growing teeth.

关键词： continuously growing teeth molar phenotype mutation stem cell regulation voles

The intestinal epithelial response to damage

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Science China(Life sciences) 2018年第10期61卷 1205-1211页

作者： Laura Weichselbaum Ophir D.Klein program in craniofacial biology and department of orofacial sciences University of CaliforniaSan Francisco CA 94143USA Institute for Medical Immunology Université Libre de BruxellesGosselies 6041Belgium Laboratory of Experimental Gastroenterology Université libre de BruxellesBrussels 1070Belgium department of Pediatrics and Institute for Human Genetics University of CaliforniaSan FranciscoCA 94143 USA

The constant renewal of the intestinal epithelium is fueled by intestinal stem cells(ISCs) lying at the base of crypts, and these ISCs continuously give rise to transit-amplifying progenitor cells during homeostasis. Upon injury and loss of ISCs, the epithelium has the ability to regenerate by the dedifferentiation of progenitor cells that then regain stemness and repopulate the pool of ISCs. Epithelial cells receive cues from immune cells, mesenchymal cells and the microbiome to maintain *** review focuses on the response of the epithelium to damage and the interplay between the different intestinal compartments.

关键词： intestine regeneration microenvironment

Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

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Cell Research 2005年第8期15卷 567-575页

作者： Paul C. SEHNKE,Beth J. LAUGHNER,Carla R. LYERLY LINEBARGER,William B. GURLEY,Robert J. FERL program in Plant Cellular and Molecular biology department of Horticultural sciences University of Florida Gainesville FL USA 32611 program in Plant Cellular and Molecular biology department of Horticultural sciences University of Florida Gainesville FL USA 32611 program in Plant Cellular and Molecular biology department of Horticultural sciences University of Florida Gainesville FL USA 32611 department of Microbiology and Cell Science University of Florida Gainesville FL USA 32611 program in Plant Cellular and Molecular biology department of Horticultural sciences University of Florida Gainesville FL USA 32611

Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs). The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood. In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A. thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

关键词： G-box binding factor interacting protein nuclear chaperone DNA binding affinity.

Meeting report:a hard look at the state of enamel research

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International Journal of Oral Science 2017年第4期9卷 193-199页

作者： ophir d klein olivier duverger wendy shaw rodrigo s lacruz derk joester janet moradian-oldak megan k pugach j timothy wright sarah e millar ashok b kulkarni john d bartlett thomas gh diekwisch pamela den besten james p simmer program in craniofacial biology and department of orofacial sciences university of californiasan franciscoSan FranciscoUSA department of pediatrics and institute for human genetics university of californiasan franciscoSan FranciscoUSA laboratory of skin biology national institute of arthritis and muscular and skin diseasesnational institutes of healthBethesdaUSA physical science division pacific northwest national laboratoriesRichlandUSA department of basic science and craniofacial biology new york university college of dentistryNewYorkUSA department of materials science and engineering northwestern universityevanstonusaEvanstonUSA center for craniofacial molecular biology herman ostrow school of dentistryuniversity of southern californiaLos AngelesUSA the forsyth institute CambridgeUSA department of developmental biology harvard school of dental medicineBostonUSA department of pediatric dentistry university of north caroline school of dentistryChapel HillUSA departments of dermatology and cel&developmental biology perelman school of medicineand department of anatomy&cell biologyschool of dental medicineuniversity of pennsylvaniaPhiladelphiaUSA functional genomics section national institute of dental and craniofacial researchBethesdaUSA division of biosciences ohio state universitycollege of dentistryColumbusUSA department of periodontics and center for craniofacial research and diagnosis texas a&m university college of dentistryDallasUSA center for children’s oral health research and department of orofacial sciences university of californiasan franciscoSan FranciscoUSA biologic and materials sciences and division of prosthodontics university of michiganAnn ArborUSA

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines （ENAMEL） Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National institute of Dental and craniofacial Research （NIDCR）. Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

关键词： enamel mineralized tissue mineralization ameloblast stem cell

Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice

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International Journal of Oral Science 2015年第2期7卷 89-94页

作者： Er-Xia Du Xiao-Fang Wang Wu-Chen Yang Deborah Kaback Siu-Pok Yee Chun-Lin Qin Anne George Jian-Jun Hao Division of Orthodontics Department of Craniofacial SciencesSchool of Dental MedicineUniversity of Connecticut department of Biomedical sciences Texas A&M Health Science CenterBaylor College of Dentistry department of Genetics and Developmental biology University of Connecticut Health Center department of Oral biology University of Illinois

Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone *** of the Fam20 C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout ***,control and regulation of the expression of Fam20 C are still *** this study,we generated a transgenic reporter model which expresses green fluorescence protein（GFP） driven by the Fam20 C *** was used to insert a 16 kb fragment of the mouse Fam20 C gene（containing the 15 kb promoter and 1.1 kb of exon 1） intoa pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation *** expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult *** was evident in the bone and teeth as early as *** GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after *** expression of GFP was significantly reduced in teeth,alveolar bone and muscle by 8 weeks of *** also observed colocalization of the GFP signal with the Fam20 C antibody in postnatal 1- and 7-day-old *** generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20 C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

关键词： transgenic promoter pBluescript osteogenesis teeth transgene reporter Fluorescence insert alveolar

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral

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International Journal of Oral Science 2023年第4期15卷 676-690页

作者： Hyun-Yi Kim Victoria Cooley Eun-Jung Kim Shujin Li Jong-Min Lee Dina Sheyfer Wenjun Liu Ophir D.Klein Derk Joester Han-Sung Jung NGeneS Inc. Ansan-siGyeonggi-doKorea department of Materials Science and Engineering Northwestern UniversityEvanstonILUSA X-ray Science Division Advanced Photon SourceArgonne National LaboratoryLemontILUSA Division in Anatomy and Developmental biology Department of Oral BiologyOral Science Research CenterBK21 FOUR ProjectYonsei University College of DentistrySeoulKorea department of orofacial sciences and program in craniofacial biology University of CaliforniaSan FranciscoCAUSA department of Pediatrics Cedars-Sinai Guerin Children’sLos AngelesCAUSA

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel,a hierarchically structured tissue comprised of highly elongated hydroxylapatite(OHAp)*** unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet,partly due to limitations with in vitro experimental ***,we demonstrate the ability to generate mineralizing dental epithelial organoids(DEOs)from adult dental epithelial stem cells(aDESCs)isolated from mouse incisor *** expressed ameloblast markers,could be maintained for more than five months(11 passages)in vitro in media containing modulators of Wnt,Egf,Bmp,Fgf and Notch signaling pathways,and were amenable to *** transplanted underneath murine kidney capsules,organoids produced OHAp crystallites similar in composition,size,and shape to mineralized dental tissues,including some enamel-like elongated *** are thus a powerful in vitro model to study mineralization process by dental epithelium,which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

关键词： epithelium maintained specialized

Genetic Analyses of Meiotic Recombination in Arabidopsis

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Journal of Integrative Plant biology 2007年第8期49卷 1199-1207页

作者： Asela J.Wijeratne Intercollege Graduate program in Plant biology Department of Biology and the Huck Institutes of the Life SciencesThe Pennsylvania State UniversityUniversity ParkPA 16802USA

Meiosis is essential for sexual reproduction and recombination is a critical step required for normal meiosis. Understanding the underlying molecular mechanisms that regulate recombination is important for medical, agricultural and ecological reasons. Readily available molecular and cytological tools make Arabidopsis an excellent system to study meiosis. Here we review recent developments in molecular genetic analyses on meiotic recombination. These include studies on plant homologs of yeast and animal genes, as well as novel genes that were first identified in plants. The characterizations of these genes have demonstrated essential functions from the initiation of recombination by double-strand breaks to repair of such breaks, from the formation of doubie-HoUiday junctions to possible resolution of these junctions, both of which are critical for crossover formation. The recent advances have ushered a new era in plant meiosis, in which the combination of genetics, genomics, and molecular cytology can uncover important gene functions.

关键词： Arabidopsis crossover formation double-Holliday junction resolution double-strand break repair model interference meioticrecombination

Genome-wide Analysis of Kelch Repeatcontaining F-box Family

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Journal of Integrative Plant biology 2007年第6期49卷 940-952页

作者： Yujin Sun Xiaofan Zhou Hong Ma department of biology and the Huck Institutes of Life sciences Intercollege Graduate program in Cell and Developmental biology The Pennsylvania State University University Park PA 16802 USA

The ubiquitin-dependent protein degradation pathway plays diverse roles in eukaryotes. Previous studies indicate that both F-box and Kelch motifs are common in a variety of organisms. F-box proteins are subunits of E3 ubiquitin ligase complexes called SCFs （SKP1, Cullinl, F-box protein, and Rbxl）; they have an N-terminal F-box motif that binds to SKP1 （S-phase kinase associated protein）, and often have C-terminal protein-protein interaction domains, which specify the protein substrates for degradation via the ubiquitin pathway. One of the most frequently found protein interaction domains in F-box proteins is the Kelch repeat domain. Although both the F-box and Kelch repeats are ancient motifs, Kelch repeats-containing F-box proteins （KFB） have only been reported for human and Arabidopsis previously. The recent sequencing of the rice genome and other plant genomes provides an opportunity to examine the possible evolution history of KFB. We carried out extensive BLAST searches to identify putative KFBs in selected organisms, and analyzed their relationships phylogenetically. We also carried out the analysis of both gene duplication and gene expression of the KFBs in rice and Arabidopsis. Our study indicates that the origin of KFBs occurs before the divergence of animals and plants, and plant KFBs underwent rapid gene duplications.

关键词： Arabidopsis thaliana F-box motif Kelch repeats Kelch repeats-containing F-box proteins SCF SKP1 like.