Single-cell sequencing technology in diabetic wound healing:New insights into the progenitors-based repair strategies

Diabetes mellitus(DM),an increasingly prevalent chronic metabolic disease,is characterised by prolonged hyperglycaemia,which leads to long-term health consequences.Although much effort has been put into understanding the pathogenesis of diabetic wounds,the underlying mechanisms remain unclear.The advent of single-cell RNA sequencing(scRNAseq)has revolutionised biological research by enabling the identification of novel cell types,the discovery of cellular markers,the analysis of gene expression patterns and the prediction of develop-mental trajectories.This powerful tool allows for an in-depth exploration of pathogenesis at the cellular and molecular levels.In this editorial,we focus on progenitor-based repair strategies for diabetic wound healing as revealed by scRNAseq and highlight the biological behaviour of various healing-related cells and the alteration of signalling pathways in the process of diabetic wound healing.ScRNAseq could not only deepen our understanding of the complex biology of diabetic wounds but also identify and validate new targets for inter-vention,offering hope for improved patient outcomes in the management of this challenging complication of DM.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

BACKGROUND The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer(CRC),one of the most prevalent malignancies worldwide.In this study,multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC.By revealing the heterogeneity and functional differences of B cells in cancer immunity,we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies.AIM To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC,explore the potential driving mechanism of B cell development,analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules,and search for possible regulatory pathways to promote the anti-tumor effects of B cells.METHODS A total of 69 paracancer(normal),tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database(https://portal.gdc.cancer.gov/).After the immune cells were sorted by multicolor flow cytometry,the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform,and the data were analyzed using bioinformatics tools such as Seurat.The differences in the number and function of B cell infiltration between tumor and normal tissue,the interaction between B cell subsets and T cells and myeloid cell subsets,and the transcription factor regulatory network of B cell subsets were explored and analyzed.RESULTS Compared with normal tissue,the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly.Among them,germinal center B cells(GCB)played the most prominent role,with positive clone expansion and heavy chain mutation level increasing,and the trend of differentiation into memory B cells increased.However,the number of plasma cells in the tumor microenvironment decreased significantly,and the plasma cells secreting IgA antibodies decreased most obviously.In addition,compared with the immune microenvironment of normal tissues,GCB cells in tumor tissues became more closely connected with other immune cells such as T cells,and communication molecules that positively regulate immune function were significantly enriched.CONCLUSION The role of GCB in CRC tumor microenvironment is greatly enhanced,and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level.Meanwhile,GCB has enhanced its association with immune cells in the microenvironment,which plays a positive anti-tumor effect.

Food nutrition and toxicology targeting on specific organs in the era of single-cell sequencing

Due to the complex natures of dietary food components,it is difficult to elucidate how the compounds affect host health.Dietary food often selectively presents its mechanism of action on different cell types,and participates in the modulation of targeted cells and their microenvironments within organs.However,the limitations of traditional in vitro assays or in vivo animal experiments cannot comprehensively examine cellular heterogeneity and the tissue-biased influences.Single-cell RNA sequencing(sc RNA-seq)has emerged as an indispensable methodology to decompose tissues into different cell types for the demonstration of transcriptional profiles of individual cells.Sc RNA-seq applications has been summarized on three typical organs(brain,liver,kidney),and two representative immune-and tumor related health problems.The everincreasing role of sc RNA-seq in dietary food research with further improvement can provide sub-cellular information and the coupling between other cellular modalities.In this review,we propose utilizing sc RNAseq to more effectively capture the subtle and complex effects of food chemicals,and how they may lead to health problems at single-cell resolution.This novel technique will be valuable to elucidate the underlying mechanism of both the health benefits of food nutrients and the detrimental consequences food toxicants at the cellular level.

Single-cell sequencing analysis reveals the molecular mechanism of promotion of SCAP proliferation upon AZD2858 treatment

The Wnt/β-catenin signaling pathway is the main target of tooth regeneration regulation.Treatment of cells with AZD2858 stimulates the Wnt/β-catenin signaling pathway,yet the function of this pathway in tooth regeneration remains unclear.Here,we found that AZD2858 promotes the accumulation ofβ-catenin in the nuclei of stem cells from the apical papilla(SCAPs)and enhances cell proliferation.Single-cell sequencing was performed on SCAPs treated with AZD2858.Eight clusters were identified,namely SCAPs-CNTNAP2,SCAPs-DTL,SCAPs-MYH11,SCAPs-MKI67,SCAPs-CXCL8,SCAPs-TPM2,SCAPs-IFIT2 and SCAPs-NEK10.The pseudo-time trajectory analysis showed that AZD2858 enhanced the evolution of SCAPs from SCAPs-TMP2 clusters to SCAPs-MYH11,SCAPs-CNTNAPs and SCAPs-NEK10 clusters via up-regulation of PRKCA,SMURF2,MAGI2,RBMS3,EXT1,CAMK2D,PLCB4,and PLCB1.These results demonstrate that AZD2858 enhances the proliferation of SCAPs-TPM2 cluster by activating the non-canonical Wnt/β-catenin signaling pathway.

Prognostic-related genes for pancreatic cancer typing and immunotherapy response prediction based on single-cell sequencing data and bulk sequencing data

Pancreatic cancer is associated with high mortality and is one of the most aggressive of malignancies,but studies have not fully evaluated its molecular subtypes,prognosis and response to immunotherapy of different subtypes.The purpose of this study was to explore the molecular subtypes and the key genes associated with the prognosis of pancreas cancer patients and study the clinical phenotype,prognosis and response to immunotherapy using single-cell seq data and bulk RNA seq data,and data retrieved from GEO and TCGA databases.Methods:Single-cell seq data and bioinformatics methods were used in this study.Pancreatic cancer data were retrieved from GEO and TCGA databases,the molecular subtypes of pancreatic cancer were determined using the six cGAS-STING related pathways,and the clinical phenotype,mutation,immunological characteristics and pathways related to pancreatic cancer were evaluated.Results:Pancreatic cancer was classified into 3 molecular subtypes,and survival analysis revealed that patients in Cluster3(C3)had the worst prognosis,whereas Cluster1(C1)had the best prognosis.The clinical phenotype and gene mutation were statistically different among the three molecular subtypes.Analysis of immunotherapy response revealed that most immune checkpoint genes were differentially expressed in the three subtypes.A lower risk of immune escape was observed in Cluster1(C1),indicating higher sensitivity to immunotherapeutic drugs and subjects in this Cluster are more likely to benefit from immunotherapy.The pathways related to pancreatic cancer were differentially enriched among the three subtypes.Five genes,namely SFRP1,GIPR,EMP1,COL17A and CXCL11 were selected to construct a prognostic signature.Conclusions:Single-cell seq data were to classify pancreatic cancer into three molecular subtypes based on differences in clinical phenotype,mutation,immune characteristics and differentially enriched pathways.Five prognosis-related genes were identified for prediction of survival of pancreatic cancer patients and to evaluate the efficacy of immunotherapy in various subtypes.

Synergy of single-cell sequencing analyses and in vivo lineage-tracing approaches:A new opportunity for stem cell biology

Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of cell surface markers and transcription factors.However,it has been challenging to define micro-heterogeneity within“stem cell”populations,each of which stands at a different level of differentiation.As stem cells become defined at a single-cell level,their differentiation path becomes clearly defined.Here,this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches,with an emphasis on practical considerations in stem cell biology.

Advances in single-cell sequencing:insights from organ transplantation

Single-cell RNA sequencing(scRNA-seq)is a comprehensive technical tool to analyze intracellular and intercellular interaction data by whole transcriptional profile analysis.Here,we describe the application in biomedical research,focusing on the immune system during organ transplantation and rejection.Unlike conventional transcriptome analysis,this method provides a full map of multiple cell populations in one specific tissue and presents a dynamic and transient unbiased method to explore the progression of allograft dysfunction,starting from the stress response to final graft failure.This promising sequencing technology remarkably improves individualized organ rejection treatment by identifying decisive cellular subgroups and cell-specific interactions.

The role of bone marrow-derived cells in the origin of liver cancer revealed by single-cell sequencing

Objective:Epithelial cancers often originate from progenitor cells,while the origin of hepatocellular carcinoma(HCC)is still controversial.HCC,one of the deadliest cancers,is closely linked with liver injuries and chronic inflammation,which trigger massive infiltration of bone marrow-derived cells(BMDCs)during liver repair.Methods:To address the possible roles of BMDCs in HCC origination,we established a diethylnitrosamine(DEN)-induced HCC model in bone marrow transplanted mice.Immunohistochemistry and frozen tissue immunofluorescence were used to verify DENinduced HCC in the pathology of the disease.The cellular origin of DEN-induced HCC was further studied by single cell sequencing,single-cell nested PCR,and immunofluorescence-fluorescence in situ hybridization.Results:Studies by using single cell sequencing and biochemical analysis revealed that HCC cells in these mice were coming from donor mice BMDCs,and not from recipient mice.Furthermore,the copy numbers of mouse orthologs of several HCC-related genes previously reported in human HCC were also altered in our mouse model.DEN-induced HCCs exhibited a similar histological phenotype and genomic profile as human HCCs.Conclusions:These results suggested that BMDCs are an important origin of HCC,which provide important clues to HCC prevention,detection,and treatments.

Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure

High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases,yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown.Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel(Healaflow■).Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure.Our results identified a total of 12 cell types,namely retinal pigment epithelial cells,rod-photoreceptor cells,bipolar cells,Müller cells,microglia,cone-photoreceptor cells,retinal ganglion cells,endothelial cells,retinal progenitor cells,oligodendrocytes,pericytes,and fibroblasts.The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells,with ganglion cells decreased by 23%.Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure.We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression.We found upregulation of the B3gat2 gene,which is associated with neuronal migration and adhesion,and downregulation of the Tsc22d gene,which participates in inhibition of inflammation.This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure.These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

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.

Single-cell sequencing analysis of the maternal-fetal interface

The microenvironment at the maternal-fetal interface is optimized to facilitate the development and survival of the fetus during pregnancy.It involves a balance between cell populations and interactions of the fetal placenta with various cell types(ie,stromal cells,endothelial cells,immune cells,and fibroblasts)that are embedded in the maternal endometrium/decidua.Aberrant shifts in cell populations and deranged cell-cell interactions are closely related to pregnancy disorders.Thus,analysis of the dynamic changes in cell populations and their interactions at the maternal-fetal interface in normal and complicated pregnancies is essential to provide insights into the fundamental processes involved in the establishment and maintenance of normal pregnancy,and how these processes are dysregulated.Thus,informing novel pathways for therapeutic targets of pregnancy complications.Single-cell sequencing(SCS)is a powerful tool for transcriptome analysis at single-cell resolution.Combined with information on the developmental trajectory and function of different cell populations,SCS can provide an unparalleled opportunity for refining the spatiotemporal cell atlas to elaborate dynamic changes in cell populations and their interactions in tissues that consist of highly heterogeneous cell populations such as the maternal-fetal interface.This minireview briefly summarizes traditional methods and their limitations for analyzing maternal-fetal interface cell-cell interactions,and introduces the current applications,advantages,limitations,and prospective applications of SCS in research on maternal-fetal interactions.

Recent developments in application of single-cell RNA sequencing in the tumour immune microenvironment and cancer therapy

The advent of single-cell RNA sequencing(scRNA-seq)has provided insight into the tumour immune microenvironment(TIME).This review focuses on the application of scRNA-seq in investigation of the TIME.Over time,scRNA-seq methods have evolved,and components of the TIME have been deciphered with high resolution.In this review,we first introduced the principle of scRNA-seq and compared different sequencing approaches.Novel cell types in the TIME,a continuous transitional state,and mutual intercommunication among TIME components present potential targets for prognosis prediction and treatment in cancer.Thus,we concluded novel cell clusters of cancerassociated fibroblasts(CAFs),T cells,tumour-associated macrophages(TAMs)and dendritic cells(DCs)discovered after the application of scRNA-seq in TIME.We also proposed the development of TAMs and exhausted T cells,as well as the possible targets to interrupt the process.In addition,the therapeutic interventions based on cellular interactions in TIME were also summarized.For decades,quantification of the TIME components has been adopted in clinical practice to predict patient survival and response to therapy and is expected to play an important role in the precise treatment of cancer.Summarizing the current findings,we believe that advances in technology and wide application of single-cell analysis can lead to the discovery of novel perspectives on cancer therapy,which can subsequently be implemented in the clinic.Finally,we propose some future directions in the field of TIME studies that can be aided by scRNA-seq technology.

Application of single-cell RNA sequencing in head and neck squamous cell carcinoma

Single-cell RNA sequencing has been broadly applied to head and neck squamous cell carcinoma(HNSCC) for characterizing the heterogeneity and genomic mutations of HNSCC benefiting from the advantage of single-cell resolution. We summarized most of the current studies and aimed to explore their research methods and ideas, as well as how to transform them into clinical applications. Through single-cell RNA sequencing, we found the differences in tumor cells’ expression programs and differentiation tracks. The studies of immune microenvironment allowed us to distinguish immune cell subpopulations, the extensive expression of immune checkpoints, and the complex crosstalk network between immune cells and non-immune cells. For cancerassociated fibroblasts(CAFs), single-cell RNA sequencing had made an irreplaceable contribution to the exploration of their differentiation status, specific CAFs markers, and the interaction with tumor cells and immune cells. In addition, we demonstrated in detail how single-cell RNA sequencing explored the HNSCC epithelial-tomesenchymal transition(EMT) model and the mechanism of drug resistance, as well as its clinical value.

Single-cell RNA sequencing reveals the dynamics of hepatic non-parenchymal cells in autoprotection against acetaminophen-induced hepatotoxicity

Gaining a better understanding of autoprotection against drug-induced liver injury(DILI)may provide new strategies for its prevention and therapy.However,little is known about the underlying mechanisms of this phenomenon.We used single-cell RNA sequencing to characterize the dynamics and functions of hepatic non-parenchymal cells(NPCs)in autoprotection against DILI,using acetaminophen(APAP)as a model drug.Autoprotection was modeled through pretreatment with a mildly hepatotoxic dose of APAP in mice,followed by a higher dose in a secondary challenge.NPC subsets and dynamic changes were identified in the APAP(hepatotoxicity-sensitive)and APAP-resistant(hepatotoxicity-resistant)groups.A chemokine(C-C motif)ligand 2^(+)endothelial cell subset almost disappeared in the APAP-resistant group,and an R-spondin 3^(+)endothelial cell subset promoted hepatocyte proliferation and played an important role in APAP autoprotection.Moreover,the dendritic cell subset DC-3 may protect the liver from APAP hepatotoxicity by inducing low reactivity and suppressing the autoimmune response and occurrence of inflammation.DC-3 cells also promoted angiogenesis through crosstalk with endothelial cells via vascular endothelial growth factor-associated ligand-receptor pairs and facilitated liver tissue repair in the APAP-resistant group.In addition,the natural killer cell subsets NK-3 and NK-4 and the Sca-1^(-)CD62L^(+)natural killer T cell subset may promote autoprotection through interferon-γ-dependent pathways.Furthermore,macrophage and neutrophil subpopulations with anti-inflammatory phenotypes promoted tolerance to APAP hepatotoxicity.Overall,this study reveals the dynamics of NPCs in the resistance to APAP hepatotoxicity and provides novel insights into the mechanism of autoprotection against DILI at a high resolution.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.

Clinical implications of single cell sequencing for bladder cancer

Bladder cancer(BC)is the 10th most common cancer worldwide,with about 0.5 million reported new cases and about 0.2 million deaths per year.In this scoping review,we summarize the current evidence regarding the clinical implications of single-cell sequencing for bladder cancer based on PRISMA guidelines.We searched PubMed,CENTRAL,Embase,and supplemented with manual searches through the Scopus,and Web of Science for published studies until February 2023.We included original studies that used at least one single-cell technology to study bladder cancer.Forty-one publications were included in the review.Twenty-nine studies showed that this technology can identify cell subtypes in the tumor microenvironment that may predict prognosis or response to immune checkpoint inhibition therapy.Two studies were able to diagnose BC by identifying neoplastic cells through single-cell sequencing urine samples.The remaining studies were mainly a preclinical exploration of tumor microenvironment at single cell level.Single-cell sequencing technology can discriminate heterogeneity in bladder tumor cells and determine the key molecular properties that can lead to the discovery of novel perspectives on cancer management.This nascent tool can advance the early diagnosis,prognosis judgment,and targeted therapy of bladder cancer.

Combining single-cell RNA-sequencing and bulk data to reveal immunity-related genes expression pattern in the systemic lupus erythematosus and target organ kidney

Background:Systemic lupus erythematosus(SLE)is a complex chronic autoimmune disease with no known cure.However,the regulatory mechanism of immunity-related genes is not fully understood in SLE.In order to explore new therapeutic targets,we used bioinformatical methods to analyze a series of data.Methods:After downloading and processing the data from Gene Expression Omnibus database,the differentially expressed genes of SLE were analyzed.CIBERSORT algorithm was used to analyze the immune infiltration of SLE.Based on single-cell RNA-sequencing data,the role of immune-related genes in SLE and its target organ(kidney)were analyzed.Key transcription factors affecting immune-related genes were identified.Cell-cell communication networks in SLE were analyzed.Results:In total,15 hub genes and 4 transcription factors were found in the bulk data.Monocytes and macrophages in GSE81622(SLE)showed more infiltration.There were four cell types were annotated in scRNA sequencing dataset(GSE135779),as follows T cells,monocyte,NK cells and B cells.Immunity-related genes were overexpressed in monocytes.Conclusion:The present study shows that immune-related genes affect SLE through monocytes and play an important role in target organ renal injury.

Single-cell RNA sequencing of meiocytes and microspores reveals the involvement of the Rf4 gene in redox homeostasis of CMS-C maize

Normal microsporogenesis is determined by both nuclear and mitochondrial genes. In maize C-type cytoplasmic male sterility, it is unclear how the development of meiocytes and microspores is affected by the mitochondrial sterility gene and the nuclear restorer gene. In this study, we sequenced the transcriptomes of single meiocytes(tetrad stage) and early mononucleate microspores from sterile and restorer lines. The numbers of expressed genes varied in individual cells and fewer than half of the expressed genes were common to the same cell types. Four comparisons revealed 3379 differentially expressed genes(DEGs), with 277 putatively associated with mitochondria, 226 encoding transcription factors,and 467 possibly targeted by RF4. KEGG analysis indicated that the DEGs in the two lines at the tetrad stage were involved predominantly in carbon metabolism and in amino acid biosynthesis and metabolism, whereas the DEGs during the transition from the tetrad stage to the early mononucleate stage were associated mostly with regulation of protein metabolism, fatty acid metabolism, and anatomical structure morphogenesis. Thus, meiocyte and microspore development was affected by the surrounding cells and the restorer gene, and the restorer gene helped restore the redox homeostasis of microspores and the normal cellular reconstruction during the transition.

Application of Single-cell Sequencing Technologies in Reproductive Medicine

We have recently witnessed the rapid development of various single-cell sequencing technologies.Pioneering single cell assays are now available for the profiling of genome,epigenome,and transcriptome.Single-cell sequencing technologies provide multi-dimensional information on early embryonic development in humans and in clinical contexts where specimens are scarce.Here,we review several available techniques and their applications in reproductive medicine.Continuing development of multimodal single-cell sequencing measurement techniques in combination with multi-omics assays will provide comprehensive profiles of an individual cell and lead to the targeted therapy for reproductive disorders and infertility.

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.