Single-cell and spatial omics:exploring hypothalamic heterogeneity

Elucidating the complex dynamic cellular organization in the hypothalamus is critical for understanding its role in coordinating fundamental body functions. Over the past decade, single-cell and spatial omics technologies have significantly evolved, overcoming initial technical challenges in capturing and analyzing individual cells. These high-throughput omics technologies now offer a remarkable opportunity to comprehend the complex spatiotemporal patterns of transcriptional diversity and cell-type characteristics across the entire hypothalamus. Current single-cell and single-nucleus RNA sequencing methods comprehensively quantify gene expression by exploring distinct phenotypes across various subregions of the hypothalamus. However, single-cell/single-nucleus RNA sequencing requires isolating the cell/nuclei from the tissue, potentially resulting in the loss of spatial information concerning neuronal networks. Spatial transcriptomics methods, by bypassing the cell dissociation, can elucidate the intricate spatial organization of neural networks through their imaging and sequencing technologies. In this review, we highlight the applicative value of single-cell and spatial transcriptomics in exploring the complex molecular-genetic diversity of hypothalamic cell types, driven by recent high-throughput achievements.

Single-cell transcriptomic profiling reveals ZEB1-mediated regulation in microglial subtypes and the impact of exercise on neuroinflammatory responses

Background:This study aims to identify distinct cellular subtypes within brain tissue using single-cell transcriptomic analysis,focusing on specific biomarkers that differentiate cell types and the effects of traditional and exercise therapy.Methods:Four samples were analyzed:older control(OC),older exercise(OE),younger control(YC),and younger exercise(YE).Single-cell RNA sequencing was used to distinguish cellular subtypes through their biomarker profiles.Data visualization included violin and t-SNE plots to illustrate biomarker expression across cell clusters such as oligodendrocytes,microglia,and astrocytes.Additionally,BV2 cells were exposed to amyloid-beta fragments to simulate Alzheimer’s disease,assessing the impact of exercise-induced cellular responses.Results:Distinct cellular subtypes were identified:oligodendrocytes(MBP,St18),microglia(Dock8),and astrocytes(Aqp4,Gpc5).Sample OE was predominantly oligodendrocytes,while YE had more astrocytes,inhibitory neurons,and Canal-Retzius cells.YC showed a significant presence of Olfm3+ganglion neurons.ZEB1 gene knockout revealed changes in SMAD family gene expression,which regulate ferroptosis.Oxidative stress levels were also evaluated.Conclusion:This profiling enhances our understanding of brain cellular functions and interactions,potentially informing targeted therapies in neurological research.Exercise may influence brain cell immune responses and cell death pathways by regulating specific gene expressions,offering new insights for treating neuroinflammation and degeneration.

Landscape of four different stages of human gastric cancer revealed by single-cell sequencing

BACKGROUND Gastric cancer(GC)poses a substantial risk to human health due to its high prevalence and mortality rates.Nevertheless,current therapeutic strategies remain insufficient.Single-cell RNA sequencing(scRNA-seq)offers the potential to provide comprehensive insights into GC pathogenesis.AIM To explore the distribution and dynamic changes of cell populations in the GC tumor microenvironment using scRNA-seq techniques.METHODS Cancerous tissues and paracancerous tissues were obtained from patients diagnosed with GC at various stages(I,II,III,and IV).Single-cell suspensions were prepared and analyzed using scRNA-seq to examine transcriptome profiles and cell-cell interactions.Additionally,quantitative real-time polymerase chain reaction(qRT-PCR)and flow cytometry were applied for measuring the expression of cluster of differentiation(CD)2,CD3D,CD3E,cytokeratin 19,cytokeratin 8,and epithelial cell adhesion molecules.RESULTS Transcriptome data from 73645 single cells across eight tissues of four patients were categorized into 25 distinct cell clusters,representing 10 different cell types.Variations were observed in these cell type distribution.The adjacent epithelial cells in stages II and III exhibited a degenerative trend.Additionally,the quantity of CD4 T cells and CD8 T cells were evidently elevated in cancerous tissues.Interaction analysis displayed a remarkable increase in interaction between B cells and other mast cells in stages II,III,and IV of GC.These findings were further validated through qRT-PCR and flow cytometry,demonstrating elevated T cells and declined epithelial cells within the cancerous tissues.CONCLUSION This study provides a comprehensive analysis of cell dynamics across GC stages,highlighting key interactions within the tumor microenvironment.These findings offer valuable insights for developing novel therapeutic strategies.

An integrated physiology and proteomics analysis reveals the response of wheat grain to low temperature stress during booting

Low temperature(LT)in spring has become one of the principal abiotic stresses that restrict the growth and development of wheat.Diverse analyses were performed to investigate the mechanism underlying the response of wheat grain development to LT stress during booting.These included morphological observation,measurements of starch synthase activity,and determination of amylose and amylopectin content of wheat grain after exposure to treatment with LT during booting.Additionally,proteomic analysis was performed using tandem mass tags(TMT).Results showed that the plumpness of wheat grains decreased after LT stress.Moreover,the activities of sucrose synthase(SuS,EC 2.4.1.13)and ADP-glucose pyrophosphorylase(AGPase,EC 2.7.7.27)exhibited a significant reduction,leading to a significant reduction in the contents of amylose and amylopectin.A total of 509 differentially expressed proteins(DEPs)were identified by proteomics analysis.The Gene Ontology(GO)enrichment analysis showed that the protein difference multiple in the nutritional repository activity was the largest among the molecular functions,and the up-regulated seed storage protein(ssP)played an active role in the response of grains to LT stress and subsequent damage.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that LT stress reduced the expression of DEPs such as sucrose phosphate synthase(SPS),glucose-1-phosphate adenylyltransferase(glgC),andβ-fructofuranosidase(FFase)in sucrose and starch metabolic pathways,thus affecting the synthesis of grain starch.In addition,many heat shock proteins(HsPs)were found in the protein processing in endoplasmic reticulum pathways,which can resist some damage caused by LT stress.These findings provide a new theoretical foundation for elucidating the underlying mechanism governing wheat yield developmentafterexposuretoLTstress inspring.

TMT-based quantitative proteomics reveals the potential mechanism of the FufangMuji Granules in carbon tetrachloride-induced liver injury

Background:Fufang Muji Granules is a traditional Chinese medicine of the Manchu ethnic group and is thought to treat hepatitis and liver injury by inhibiting the elevation of alpha-fetoprotein.Methods:In this investigation,tandem mass tag(TMT)-based quantitative proteomics was performed to figure out the therapeutic mechanisms of Fufang Muji Granules on liver injury caused by carbon tetrachloride(CCl_(4))in rats.Results:Biochemical analyses(alanine aminotransferase;glutamate aminotransferase;aspartate aminotransferase)and histologic analyses(hematoxylin-eosin)demonstrated that FMG was effective in ameliorating liver injury.A sum of 6,208 proteins were identified and 2,475 proteins were determined as differential abundance proteins(DAPs)in rat liver treated with Fufang Muji Granules which compared to the model group.Bioinformatics analysis indicated that the DAPs are primarily enriched in multiple pathways such as rno00280(valine,leucine,and isoleucine degradation),rno00640(Propanoate metabolism),and rno00380(Tryptophan metabolism).Western blot was employed to validate the findings from the proteomic analysis.Conclusion:This study not only provides useful information on the mechanism of Fufang Muji Granules in the treatment of liver injury but also serves as a basis for further study of Fufang Muji Granules in vivo.

Transcriptome and single-cell profiling of the mechanism of diabetic kidney disease

BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the role of the NLRP3 inflammasome in DKD.METHODS Using datasets from the Gene Expression Omnibus database,30 NLRP3 inflammasome-related genes were identified.Differentially expressed genes were selected using differential expression analysis,whereas intersecting genes were selected based on overlapping differentially expressed genes and NLRP3 inflammasome-related genes.Subsequently,three machine learning algorithms were used to screen genes,and biomarkers were identified by overlapping the genes from the three algorithms.Potential biomarkers were validated by western blotting in a db/db mouse model of diabetes.RESULTS Two biomarkers,sirtuin 2(SIRT2)and caspase 1(CASP1),involved in the Leishmania infection pathway were identified.Both biomarkers were expressed in endothelial cells.Pseudo-temporal analysis based on endothelial cells showed that DKD mostly occurs during the mid-differentiation stage.Western blotting results showed that CASP1 expression was higher in the DKD group than in the control group(P<0.05),and SIRT2 content decreased(P<0.05).CONCLUSION SIRT2 and CASP1 provide a potential theoretical basis for DKD treatment.

Single-cell transcriptomic atlas of goat ovarian aging

Background The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process,and ovarian aging is shown by a decrease in the number and quality of oocytes.However,little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging,especially in goats.Therefore,the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.Results For the first time,we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn,young and aging goats,and identified nine ovarian cell types with distinct gene-expression signatures.Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes,such as Wnt beta-catenin signalling was enriched in germ cells,whereas ovarian steroidogenesis was enriched in granulosa cells(GCs).Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system,oxidative phosphorylation,and apoptosis.Subsequently,we identified a series of dynamic genes,such as AMH,CRABP2,THBS1 and TIMP1,which determined the fate of GCs.Additionally,FOXO1,SOX4,and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.Conclusions This study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell typespecific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

Strategies for translating proteomics discoveries into drug discovery for dementia

Tauopathies,diseases characterized by neuropathological aggregates of tau including Alzheimer's disease and subtypes of fro ntotemporal dementia,make up the vast majority of dementia cases.Although there have been recent developments in tauopathy biomarkers and disease-modifying treatments,ongoing progress is required to ensure these are effective,economical,and accessible for the globally ageing population.As such,continued identification of new potential drug targets and biomarkers is critical."Big data"studies,such as proteomics,can generate information on thousands of possible new targets for dementia diagnostics and therapeutics,but currently remain underutilized due to the lack of a clear process by which targets are selected for future drug development.In this review,we discuss current tauopathy biomarkers and therapeutics,and highlight areas in need of improvement,particularly when addressing the needs of frail,comorbid and cognitively impaired populations.We highlight biomarkers which have been developed from proteomic data,and outline possible future directions in this field.We propose new criteria by which potential targets in proteomics studies can be objectively ranked as favorable for drug development,and demonstrate its application to our group's recent tau interactome dataset as an example.

Single-cell profiling of the pig cecum at various developmental stages

The gastrointestinal tract is essential for food digestion,nutrient absorption,waste elimination,and microbial defense.Single-cell transcriptome profiling of the intestinal tract has greatly enriched our understanding of cellular diversity,functional heterogeneity,and their importance in intestinal tract development and disease.Although such profiling has been extensively conducted in humans and mice,the single-cell gene expression landscape of the pig cecum remains unexplored.Here,single-cell RNA sequencing was performed on 45572 cells obtained from seven cecal samples in pigs at four different developmental stages(days(D)30,42,150,and 730).Analysis revealed 12 major cell types and 38 subtypes,as well as their distinctive genes,transcription factors,and regulons,many of which were conserved in humans.An increase in the relative proportions of CD8^(+)T and Granzyme A(low expression)natural killer T cells(GZMA^(low)NKT)cells and a decrease in the relative proportions of epithelial stem cells,Tregs,RHEX^(+)T cells,and plasmacytoid dendritic cells(pDCs)were noted across the developmental stages.Moreover,the post-weaning period exhibited an up-regulation in mitochondrial genes,COX2 and ND2,as well as genes involved in immune activation in multiple cell types.Cell-cell crosstalk analysis indicated that IBP6^(+)fibroblasts were the main signal senders at D30,whereas IBP6^(−)fibroblasts assumed this role at the other stages.NKT cells established interactions with epithelial cells and IBP6^(+)fibroblasts in the D730 cecum through mediation of GZMA-F2RL1/F2RL2 pairs.This study provides valuable insights into cellular heterogeneity and function in the pig cecum at different development stages.

Proteomics for early prenatal screening of gestational diabetes mellitus

In this editorial,we comment on the article by Cao et al.Through applying isobaric tags for relative and absolute quantification technology coupled with liquid chromatography-tandem mass spectrometry,the researchers observed significant differential expression of 47 proteins when comparing serum samples from pregnant women with gestational diabetes mellitus(GDM)to the healthy ones.GDM symptoms may involve abnormalities in inflammatory response,complement system,coagulation cascade activation,and lipid metabolism.Retinol binding protein 4 and angiopoietin like 8 are potential early indicators of GDM.GDM stands out as one of the most prevalent metabolic complications during pregnancy and is linked to severe maternal and fetal outcomes like pre-eclampsia and stillbirth.Nevertheless,none of the biomarkers discovered so far have demonstrated effectiveness in predicting GDM.Our topic was designed to foster insights into advances in the application of proteomics for early prenatal screening of GDM.

Identification and validation of a pyroptosis-related prognostic model for colorectal cancer based on bulk and single-cell RNA sequencing data

BACKGROUND Pyroptosis impacts the development of malignant tumors,yet its role in colorectal cancer(CRC)prognosis remains uncertain.AIM To assess the prognostic significance of pyroptosis-related genes and their association with CRC immune infiltration.METHODS Gene expression data were obtained from The Cancer Genome Atlas(TCGA)and single-cell RNA sequencing dataset GSE178341 from the Gene Expression Omnibus(GEO).Pyroptosis-related gene expression in cell clusters was analyzed,and enrichment analysis was conducted.A pyroptosis-related risk model was developed using the LASSO regression algorithm,with prediction accuracy assessed through K-M and receiver operating characteristic analyses.A nomo-gram predicting survival was created,and the correlation between the risk model and immune infiltration was analyzed using CIBERSORTx calculations.Finally,the differential expression of the 8 prognostic genes between CRC and normal samples was verified by analyzing TCGA-COADREAD data from the UCSC database.RESULTS An effective pyroptosis-related risk model was constructed using 8 genes-CHMP2B,SDHB,BST2,UBE2D2,GJA1,AIM2,PDCD6IP,and SEZ6L2(P<0.05).Seven of these genes exhibited differential expression between CRC and normal samples based on TCGA database analysis(P<0.05).Patients with higher risk scores demonstrated increased death risk and reduced overall survival(P<0.05).Significant differences in immune infiltration were observed between low-and high-risk groups,correlating with pyroptosis-related gene expression.CONCLUSION We developed a pyroptosis-related prognostic model for CRC,affirming its correlation with immune infiltration.This model may prove useful for CRC prognostic evaluation.

Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics

BACKGROUND As a well-known fact to the public,gestational diabetes mellitus(GDM)could bring serious risks for both pregnant women and infants.During this important investigation into the linkage between GDM patients and their altered expression in the serum,proteomics techniques were deployed to detect the differentially expressed proteins(DEPs)of in the serum of GDM patients to further explore its pathogenesis,and find out possible biomarkers to forecast GDM occurrence.METHODS Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria.Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation,and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry.Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis,and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA).RESULTS Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDMgravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteinsassociated with lipid metabolism, coagulation cascade activation, complement system and inflammatory responsein the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serumof GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk ofgestation.CONCLUSION GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complementsystem and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

The early life immune dynamics and cellular drivers at single-cell resolution in lamb forestomachs and abomasum

Background Four-chambered stomach including the forestomachs(rumen,reticulum,and omasum)and abomasum allows ruminants convert plant fiber into high-quality animal products.The early development of this four-chambered stomach is crucial for the health and well-being of young ruminants,especially the immune development.However,the dynamics of immune development are poorly understood.Results We investigated the early gene expression patterns across the four-chambered stomach in Hu sheep,at 5,10,15,and 25 days of age.We found that forestomachs share similar gene expression patterns,all four stomachs underwent widespread activation of both innate and adaptive immune responses from d 5 to 25,whereas the metabolic function were significantly downregulated with age.We constructed a cell landscape of the four-chambered stomach using single-cell sequencing.Integrating transcriptomic and single-cell transcriptomic analyses revealed that the immune-associated module hub genes were highly expressed in T cells,monocytes and macrophages,as well as the defense-associated module hub genes were highly expressed in endothelial cells in the four-stomach tissues.Moreover,the non-immune cells such as epithelial cells play key roles in immune maturation.Cell communication analysis predicted that in addition to immune cells,non-immune cells recruit immune cells through macrophage migration inhibitory factor signaling in the forestomachs.Conclusions Our results demonstrate that the immune and defense responses of four stomachs are quickly developing with age in lamb's early life.We also identified the gene expression patterns and functional cells associated with immune development.Additionally,we identified some key receptors and signaling involved in immune regulation.These results help to understand the early life immune development at single-cell resolution,which has implications to develop nutritional manipulation and health management strategies based on specific targets including key receptors and signaling pathways.

Single-cell transcriptome sequencing reveals the mechanism regulating rice plumule development

Seed plumules comprise multiple developing tissues and are key sites for above-ground plant organ morphogenesis.Here,the spatial expression of genes in developing rice seed plumules was characterized by single-cell transcriptome sequencing in Zhongjiazao 17,a popular Chinese indica rice cultivar.Of 15 cell clusters,13 were assigned to cell types using marker genes and cluster-specific genes.Marker genes of multiple cell types were expressed in several clusters,suggesting a complex developmental system.Some genes for signaling by phytohormones such as abscisic acid were highly expressed in specific clusters.Various cis-elements in the promoters of genes specifically expressed in cell clusters were calculated,and some key hormone-related motifs were frequent in certain clusters.Spatial expression patterns of genes involved in rapid seed germination,seedling growth,and development were identified.These findings enhanced our understanding of cellular diversity and specialization within plumules of rice,a monocotyledonous model crop.

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells

Objective Hydroquinone(HQ),one of the phenolic metabolites of benzene,is widely recognized as an important participant in benzene-induced hematotoxicity.However,there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn’t been fully understood yet.Methods In this study,we treated K562 cells with 40μmol/L HQ for 72 h,examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring(PRM),and performed bioinformatics analysis to identify interaction networks.Results One hundred and eighty-seven upregulated differentially expressed proteins(DEPs)and 279 downregulated DEPs were identified in HQ-exposed K562 cells,which were involved in neutrophilmediated immunity,blood microparticle,and other GO terms,as well as the lysosome,metabolic,cell cycle,and cellular senescence-related pathways.Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways,we constructed the network of protein interactions and determined 6 DEPs(STAT1,STAT3,CASP3,KIT,STAT5B,and VEGFA)as main hub proteins with the most interactions,among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity.Conclusion Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

Comparative proteomics reveals the response and adaptation mechanisms of white Hypsizygus marmoreus against the biological stress caused by Penicillium

White Hypsizygus marmoreus is a popular edible mushroom.Its mycelium is easy to be contaminated by Penicillium,which leads to a decrease in its quality and yield.Penicillium could compete for limited space and nutrients through rapid growth and produce a variety of harmful gases,such as benzene,aldehydes,phenols,etc.,to inhibit the growth of H.marmoreus mycelium.A series of changes occurred in H.marmoreus proteome after contamination when detected by the label-free tandem mass spectrometry(MS/MS)technique.Some proteins with up-regulated expression worked together to participate in some processes,such as the non-toxic transformation of harmful gases,glutathione metabolism,histone modification,nucleotide excision repair,clearing misfolded proteins,and synthesizing glutamine,which were mainly used in response to biological stress.The proteins with down-regulated expression are mainly related to the processes of ribosome function,protein processing,spliceosome,carbon metabolism,glycolysis,and gluconeogenesis.The reduction in the function of these proteins affected the production of the cell components,which might be an adjustment to adapt to growth retardation.This study further enhanced the understanding of the biological stress response and the growth restriction adaptation mechanisms in edible fungi.It also provided a theoretical basis for protein function exploration and edible mushroom food safety research.

Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation.However,effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult.Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols,which have partially addressed these challenges.In this review,we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data.Furthermore,we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells,delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis,investigating abnormal spermatogenesis in humans,and,ultimately,elucidating the molecular evolution of mammalian spermatogenesis.

Single-cell transcriptomics reveals T-cell heterogeneity and immunomodulatory role of CD4^(+) T native cells in Candida albicans infection

Objective:Candida albicans is a common fungal pathogen that triggers complex host defense mechanisms,including coordinated innate and adaptive immune responses,to neutralize invading fungi effectively.Exploring the immune microenvironment has the potential to inform the development of therapeutic strategies for fungal infections.Methods:The study analyzed individual immune cell profiles in peripheral blood mononuclear cells from Candida albicans-infected mice and healthy control mice using single-cell transcriptomics,fluorescence quantitative PCR,and Western blotting.We investigated intergroup differences in the dynamics of immune cell subpopulation infiltration,pathway enrichment,and differentiation during Candida albicans infection.Results:Our findings indicate that infiltration of CD4^(+)naive cells,regulatory T(Treg)cells,and Microtubules(MT)-associated cells increased after infection,along with impaired T cell activity.Notably,CD4^(+) T cells and plasma cells were enhanced after infection,suggesting that antibody production is dependent on T cells.In addition,we screened 6 hub genes,transcription factor forkhead box protein 3(Foxp3),cytotoxic T-lymphocyte associated protein 4(CTLA4),Interleukin 2 Receptor Subunit Beta(Il2rb),Cd28,C-C Motif Chemokine Ligand 5(Ccl5),and Cd27 for alterations associated with CD4^(+) T cell differentiation.Conclusions:These results provide a comprehensive immunological landscape of the mechanisms of Candida albicans infection and greatly advance our understanding of adaptive immunity in fungal infections.

Revolutionizing stem cell research:unbiased insights through single-cell sequencing

Stem cells have shown great application potential in wound repair,tissue regeneration,and disease treatment.Therefore,a full understanding of stem cells and their related regulatory mechanisms in disease treatment is conducive to improving the therapeutic effect of stem cells.However,thus far,there are still many unsolved mysteries in thefield of stem cells due to technical limitations,which hinder the in-depth exploration of stem cells and their wide clinical application.Single-cell sequencing(SCS)has provided very powerful and unbiased insights into cell gene expression profiles at the single-cell level,bringing exciting results to the stem cellfield.At present,SCS has been widely applied in thefield of stem cells,covering various aspects,including lineage tracing the development of stem cells,identifying new stem cell types,exploring cellular heterogeneity,and identifying internal functional subpopulations.In this paper,we focus on the latest research progress and discuss the application of SCS technology in stem cells.

Insights gained from single-cell analysis of chimeric antigen receptor T-cell immunotherapy in cancer

Advances in chimeric antigen receptor(CAR)-T cell therapy have significantly improved clinical outcomes of patients with relapsed or refractory hematologic malignancies.However,progress is still hindered as clinical benefit is only available for a fraction of patients.A lack of understanding of CAR-T cell behaviors in vivo at the single-cell level impedes their more extensive application in clinical practice.Mounting evidence suggests that single-cell sequencing techniques can help perfect the receptor design,guide gene-based T cell modification,and optimize the CAR-T manufacturing conditions,and all of them are essential for long-term immunosurveillance and more favorable clinical outcomes.The information generated by employing these methods also potentially informs our understanding of the numerous complex factors that dictate therapeutic efficacy and toxicities.In this review,we discuss the reasons why CAR-T immunotherapy fails in clinical practice and what this field has learned since the milestone of single-cell sequencing technologies.We further outline recent advances in the application of single-cell analyses in CAR-T immunotherapy.Specifically,we provide an overview of single-cell studies focusing on target antigens,CAR-transgene integration,and preclinical research and clinical applications,and then discuss how it will affect the future of CAR-T cell therapy.