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

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

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Autologous fibroblasts induce fibrosis of the nucleus pulposus to maintain the stability of degenerative intervertebral discs

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Bone Research 2020年第1期8卷 63-75页

作者： Chen Chen Tangjun Zhou Xiaojiang Sun Chen Han Kai Zhang Changqing Zhao Xunlin Li Haijun Tian Xiao Yang Yifan Zhou Zhiqian Chen An Qin Jie Zhao Shanghai Key laboratory of orthopaedic Implants Department of Orthopaedic Surgery Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine

Lumbar degenerative disc diseases cause low back pain(LBP). The maintenance of the height and stability of the intervertebral disc(IVD) space is an effective treatment for LBP. The following study evaluated the effects of fibroblast injection on intervertebral disc degeneration(IDD) in a preclinical setting. Compared with the IDD group, the fibroblast treatment group demonstrated effective maintenance of IVD height, reduced endplate degeneration, and improved nuclear magnetic resonance signals and overall histological structure. In doing so, fibrotic IVDs maintained the stability and biomechanics of the vertebra. This finding is in agreement with clinical findings that human nucleus pulposus(NP) fibrosis is essential for the maintenance of IVD height and mechanical properties in patients following percutaneous endoscopic lumbar discectomy(PELD). Mechanistically, we demonstrated that injected fibroblasts not only proliferated but also induced NP cells to adopt a fibrotic phenotype via the secretion of TGF-β.Finally, to better mimic human conditions, the efficacy of autologous fibroblast injection in the treatment of IDD was further examined in a nonhuman primate cynomolgus monkey model due to their capacity for upright posture. We showed that the injection of fibroblasts could maintain the IVD height and rescue IVD signals in cynomolgus monkeys. Taken together, the results of our study reveal that autologous fibroblast injection can enhance the natural process of fibrosis during acute and subacute stages of stress-induced IDD. Fibrotic IVDs can maintain the stability, biological activity, and mechanical properties of the intervertebral space, thus providing a new direction for the treatment of intervertebral space-derived lumbar degenerative diseases.

关键词： intervertebral degenerative degeneration

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Engineering 3D approaches to model the dynamic microenvironments of cancer bone metastasis

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Bone Research 2018年第1期6卷 38-49页

作者： Han Qiao Tingting Tang Shanghai Key laboratory of orthopaedic Implants Department of Orthopaedic Surgery Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine

Cancer metastasis to bone is a three-dimensional(3D), multistep, dynamic process that requires the sequential involvement of three microenvironments, namely, the primary tumour microenvironment, the circulation microenvironment and the bone microenvironment. Engineered 3D approaches allow for a vivid recapitulation of in vivo cancerous microenvironments in vitro, in which the biological behaviours of cancer cells can be assessed under different metastatic conditions. Therefore, modelling bone metastasis microenvironments with 3 D cultures is imperative for advancing cancer research and anti-cancer treatment strategies. In this review, multicellular tumour spheroids and bioreactors, tissue engineering constructs and scaffolds, microfluidic systems and 3D bioprinting technology are discussed to explore the progression of the 3D engineering approaches used to model the three microenvironments of bone metastasis. We aim to provide new insights into cancer biology and advance the translation of new therapies for bone metastasis.

关键词： three-dimensional anti-cancer treatment modelling bone metastasis

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Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-κB/MAPK signaling and protecting chondrocytes

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Acta Pharmaceutica Sinica B 2019年第5期9卷 973-985页

作者： Feng Zhou Jingtian Mei Xiuguo Han Hanjun Li Shengbing Yang Minqi Wang Linyang Chu Han Qiao Tingting Tang Shanghai Key laboratory of orthopaedic Implants Department of Orthopaedic Surgery Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine

The objective was to investigate the effect of kinsenoside(Kin) treatments on macrophage polarity and evaluate the resulting protection of chondrocytes to attenuate osteoarthritis(OA) ***264.7 macrophages were polarized to M1/M2 subtypes then administered with different concentrations of Kin. The polarization transitions were evaluated with quantitative real-time polymerase chain reaction(q RT-PCR), confocal observation and flow cytometry analysis. The mechanism of Kin repolarizing M1 macrophages was evaluated by Western blot. Further, macrophage conditioned medium(CM) and IL-1β were administered to chondrocytes. Micro-CT scanning and histological observations were conducted in vivo on anterior cruciate ligament transection(ACLT) mice with or without Kin treatment. We found that Kin repolarized M1 macrophages to the M2 phenotype. Mechanistically, Kin inhibited the phosphorylation of IκBα, which further reduced the downstream phosphorylation of P65 in nuclear factor-κB(NF-κB) signaling. Moreover, Kin inhibited mitogen-activated protein kinases(MAPK) signaling molecules p-JNK, p-ERK and p-P38. Additionally, Kin attenuated macrophage CM and IL-1β-induced chondrocyte damage. In vivo, Kin reduced the infiltration of M1 macrophages,promoted M2 macrophages in the synovium, inhibited subchondral bone destruction and reduced articular cartilage damage induced by ACLT. All the results indicated that Kin is an effective therapeutic candidate for OA treatment.

关键词： Kinsenoside Osteoarthritis Macrophages Polarization Chondrocytes

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In vivo bone dynamic imaging of PTH's anabolic effect

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医用生物力学 2013年第S1期28卷 47-50页

作者： Allison R.Altman Chantal de Bakker Wei-Ju Tseng Beom Kang Huh Abhishek Chandra Ling Qin X.Sherry Liu McKay orthopaedic Research laboratory Department of Orthopaedic SurgeryPereman School of MedicineUniversity of Pennsylvania

Introduction Aging shifts bone remodeling toward a negative balance between bone formation and resorption,causing bone loss and increased fracture ***-resorptive agents are commonly used to inhibit bone resorption and stabilize bone *** they are effective to prevent further bone loss,there is also a great need for anabolic agents which can reverse bone deterioration and regain lost skeletal ***,the only FDA-approved anabolic treatment for osteoporosis,greatly stimulates bone *** therapy of anti-resorptive treatments,such as alendronate(ALN),and PTH have been proposed and are expected to further increase bone ***,studies show conflicting results regarding the effectiveness

关键词： osteoporosis remodeling trabecular skeletal deterioration impaired inhibit stabilize approved histology

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Osteogenesis of Adipose-Derived Stem Cells

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Bone Research 2013年第2期1卷 133-145页

作者： Brian E. Grottkau Yunfeng Lin Department of orthopaedic Surgery MassGeneral Hospital for Children and the Pediatric Orthopaedic Laboratory for Tissue Engineering and Regenerative MedicineHarvard Medical SchoolBostonMassachusettsUSA

Current treatment options for skeletal repair, including immobilization, rigid fixation, alloplastic materials and bone grafts, have significant limitations. Bone tissue engineering offers a promising method for the repair of bone deficieny caused by fractures, bone loss and tumors. The use of adipose derived stem cells （ASCs） has received attention because of the self-renewal ability, high proliferative capacity and potential of osteogenic differentiation in vitro and in vivo studies of bone regeneration. Although cell therapies using ASCs are widely promising in various clinical fields, no large human clinical trials exist for bone tissue engineering. The aim of this review is to introduce how they are harvested, examine the characterization of ASCs, to review the mechanisms of osteogenic differentiation, to analyze the effect of mechanical and chemical stimuli on ASC osteodifferentiation, to summarize the current knowledge about usage of ASC in vivo studies and clinical trials, and finally to conclude with a general summary of the field and comments on its future direction.

关键词： adipose derived stem cells osteogenesis bone tissue engineering osteogenic differentiation

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CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFβ_3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ_1

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Chinese Medical Sciences Journal 2007年第2期22卷 113-118页

作者： Jia-ming Sai You-gu Hu De-chun Wang Department of orthopaedic Surgery the Affiliated Hospital of Medical College Qingdao University Qingdao 266003 orthopaedic laboratory Orthopaedics and Traumatology Hospital Qingdao 266003

Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1. Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site SalⅠ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plasmid AAV-TGFβ3 was transfected into H293 cells with LipofectamineTM 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ1 in the earlier and later dedifferentiated NP cells. Results For the earlier dedifferentiated NP cells, AAV-TGFβ3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ1 inhibited its synthesis. Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

关键词： adeno-associated virus transforming growth factor β3 transforming growth factor β1 nucleus pulpous proteoglycan

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Progress in the Application of 3D Printing Technology in Spine Surgery

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Journal of Shanghai Jiaotong university(Science) 2021年第3期26卷 352-360页

作者： SUN Xiaojiang YANG Erzhu ZHAO Changqing CHENG Xiaofei ZHANG Kai TIAN Haijun DING Baozhi LI Hua JIANG Wenbo DAI Kerong ZHAO Jie Department of orthopaedic Surgery Shanghai Key Laboratory of Orthopaedic ImplantsShanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghai200011China

We are in the midst of exciting advancements in new technologies and innovative research in precision *** these,3D printing is one of the most frequently seen in clinical orthopaedic *** new technique has been adopted in a vast range of applications in spine surgery,such as producing anatomical models,surgical templates,preoperative plans,and spinal *** studies on 3D printing technologies in spine surgery have reported the benefits of this emerging technology with more effective manufacturing,more visualisation for communication,and more precise navigation for screw insertion and *** addition,in customised implant design and fabrication processes,3D printing products with anatomical adaptions and complex porous microstructure show some attractive advantages in terms of fit and ***,there are still some concerns about the safety and feasibility of the application of 3D printing technology in spine *** review the literature on and share our experiences with the application of 3D printing from the beginning of collaborations between doctors and computer-aided design(CAD)designers to the final follow-up of clinical patients.

关键词： 3D printing spine patient-specific implants anatomical model template

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Comparison of Effects of Mechanical Stretching on Osteogenic Potential of ASCs and BMSCs

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Bone Research 2013年第3期1卷 282-290页

作者： Brian E. Grottkau Xingmei Yang Liang Zhang Ling Ye Yunfeng Lin Department of orthopaedic Surgery MassGeneral Hospital for Children and the Pediatric Orthopaedic Laboratory for Tissue Engineering and Regenerative Medicine Harvard Medical SchoolBoston Massachusetts USA State Key laboratory of Oral Diseases West China Hospital of Stomatology Sichuan University

Mechanical forces play critical roles in the development and remodeling processes of bone. As an alternative cell source for bone engineering, adipose-derived stem cells （ASCs） should be fully investigated for their responses to mechanical stress. Similarly, the osteogenic potential, stimulated by mechanical stress, should be compared with bone marrow stromal cells （BMSCs）, which have been clinically used for bone tissue engineering. In this study, ASCs and BMSCs were osteogenic-induced for 48 hours, and then subjected to uniaxial mechanical stretching for 2 or 6 hours. Cell orientation, osteogenic regulatory genes, osteogenic genes and ALP activities were measured and compared between ASCs and BMSCs. ASCs could align in a perpendicular way to the direction of stretching stress, while BMSCs did not present a specific alignment. Both 2 and 6 hours mechanical stretching could enhance the mRNA expression of Osx and Runx2 in BMSCs and ASCs, while OCN mRNA only increased in ASCs after 6 hours mechanical loading. Mechanical stretching enhanced the BMP-2 mRNA expression in ASCs, while only after 6 hours of mechanical loading significantly increased the BMP-2 gene expression in BMSCs. Significant differences only exist between ASCs and BMSCs loaded at 2 hours of mechanical stretching. It is concluded that ASCs are more rapid responders to mechanical stress, and have greater potential than BMSCs in osteogenesis when stimulated by mechanical stretching, indicating their usefulness for bone study in a rat model.

关键词： mechanotransduction stem cells osteoblasts adipocytes

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The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts

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Bone Research 2014年第2期2卷 110-120页

作者： Thomas S Lisse Kanagasabai Vadivel S Paul Bajaj Rui Zhou Rene F Chun Martin Hewison John S Adams Department of orthopaedic Surgery and the orthopaedic Hospital Research Center UCLA Los Angeles CA USA Department of Regenerative Biology and Medicine Mount Desert Island Biological Laboratory Bar Harbor ME USA

Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

关键词： gene The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts Figure EcoRI

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