Search

Head and neck cancer (HNC) is the sixth most common disease in the world. The recurrence rate of patients is relatively high, and the heterogeneity of tumor immune microenvironment (TIME) cells may be an important reason for this. Single-cell sequencing (SCS) is currently the most promising and mature application in cancer research. It can identify unique genes expressed in cells and study tumor heterogeneity. According to current research, the heterogeneity of immune cells has become an important factor affecting the occurrence and development of HNC. SCSs can provide effective therapeutic targets and prognostic factors for HNC patients through analyses of gene expression levels and cell heterogeneity. Therefore, this study analyzes the basic theory of HNC and the development of SCS technology, elaborating on the application of SCS technology in HNC and its potential value in identifying HNC therapeutic targets and biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2025. Published by Elsevier B.V.)

Backgrounds: Bladder cancer (BLCA) has a high degree of intratumor heterogeneity, which significantly affects patient prognosis. We performed single-cell analysis of BLCA tumors and organoids to elucidate the underlying mechanisms., Methods: Single-cell RNA sequencing (scRNA-seq) data of BLCA samples were analyzed using Seurat, harmony, and infercnv for quality control, batch correction, and identification of malignant epithelial cells. Gene set enrichment analysis (GSEA), cell trajectory analysis, cell cycle analysis, and single-cell regulatory network inference and clustering (SCENIC) analysis explored the functional heterogeneity between malignant epithelial cell subpopulations. Cellchat was used to infer intercellular communication patterns. Co-expression analysis identified co-expression modules of the anti-apoptotic subpopulation. A prognostic model was constructed using hub genes and Cox regression, and nomogram analysis was performed. The tumor immune dysfunction and exclusion (TIDE) algorithm was applied to predict immunotherapy response., Results: Organoids recapitulated the cellular and mutational landscape of the parent tumor. BLCA progression was characterized by mesenchymal features, epithelial-mesenchymal transition (EMT), immune microenvironment remodeling, and metabolic reprograming. An anti-apoptotic tumor subpopulation was identified, characterized by aberrant gene expression, transcriptional instability, and a high mutational burden. Key regulators of this subpopulation included CEBPB, EGR1, ELF3, and EZH2. This subpopulation interacted with immune and stromal cells through signaling pathways such as FGF, CXCL, and VEGF to promote tumor progression. Myofibroblast cancer-associated fibroblasts (mCAFs) and inflammatory cancer-associated fibroblasts (iCAFs) differentially contributed to metastasis. Protein-protein interaction (PPI) network analysis identified functional modules related to apoptosis, proliferation, and metabolism in the anti-apoptotic subpopulation. A 5-gene risk model was developed to predict patient prognosis, which was significantly associated with immune checkpoint gene expression, suggesting potential implications for immunotherapy., Conclusions: We identified a distinct anti-apoptotic tumor subpopulation as a key driver of tumor progression with prognostic significance, laying the foundation for the development of new therapeutic strategies to improve patient outcomes., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2025.)

Pyroptosis is a regulated form of inflammatory cell death in which Gasdermin D (GSDMD) plays a central role as the key effector molecule. GSDMD-mediated pyroptosis is characterized by complex biological features and considerable heterogeneity in its expression, mechanisms, and functional outcomes across various tissues, cell types, and pathological microenvironments. This heterogeneity is particularly pronounced in inflammation-related diseases and tumors. In the context of inflammatory diseases, GSDMD expression is typically upregulated, and its activation in macrophages, neutrophils, T cells, epithelial cells, and mitochondria triggers both pyroptotic and non-pyroptotic pathways, leading to the release of pro-inflammatory cytokines and exacerbation of tissue damage. However, under certain conditions, GSDMD-mediated pyroptosis may also serve a protective immune function. The expression of GSDMD in tumors is regulated in a more complex manner, where it can either promote immune evasion or, in some instances, induce tumor cell death. As our understanding of GSDMD's role continues to progress, there have been advancements in the development of inhibitors targeting GSDMD-mediated pyroptosis; however, these therapeutic interventions remain in the preclinical phase. This review systematically examines the cellular and molecular complexities of GSDMD-mediated pyroptosis, with a particular emphasis on its roles in inflammation-related diseases and cancer. Furthermore, it underscores the substantial therapeutic potential of GSDMD as a target for precision medicine, highlighting its promising clinical applications., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors have declared no conflict of interest., (© 2025. The Author(s).)

Glucose metabolism is a key factor characterizing the cellular state during multicellular development. In metazoans, the metabolic state of undifferentiated cells correlates with growth/differentiation transition and cell fate determination. Notably, the cell fate of the Amoebozoa species Dictyostelium discoideum is biased by the presence of glucose and is also correlated with early differences in intracellular ATP. However, the relationship between early cell-cell heterogeneity, cell differentiation, and the metabolic state is unclear. To address the link between glucose metabolism and cell differentiation in D. discoideum , we studied the role of phosphoenolpyruvate carboxylase (PEPC), a key enzyme in the PEP-oxaloacetate-pyruvate node, a core junction that dictates the metabolic flux of glycolysis, the TCA cycle, and gluconeogenesis. We demonstrate that there is cell-cell heterogeneity in PEPC promoter activity in vegetative cells, which depends on nutrient conditions, and that cells with high PEPC promoter activity differentiate into spores. The PEPC null mutant exhibited an aberrantly high prestalk/prespore ratio, and the spore mass of the fruiting body was glassy and consisted of immature spores. Furthermore, the PEPC null mutant had high ATP levels and low mitochondrial membrane potential. Our results suggest the importance of cell-cell heterogeneity in the levels of metabolic enzymes during early cell fate priming., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Abe, Hashimura, Hiraoka, Fujishiro, Kameya, Taoka, Kuwana, Fukuzawa and Sawai.)

Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance. KEY POINTS: • Strategy to study plasmid recombination during bioproduction. • Different plasmid structures can be identified and monitored by flow cytometry. • Mathematical modelling suggests specific alterations in plasmid structures., Competing Interests: Declarations. This article does not contain any studies with animals performed by any of the authors. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

To maintain and regenerate adult tissues after injury, division and differentiation of tissue-resident stem cells must be precisely regulated. It remains elusive which regulatory strategies prevent exhaustion or overgrowth of the stem cell pool, whether there is coordination between multiple mechanisms, and how to detect them from snapshots. In Drosophila testes, somatic stem cells transition to a state that licenses them to differentiate, but remain capable of returning to the niche and resuming cell division. Here, we build stochastic mathematical models for the somatic stem cell population to investigate how licensing contributes to homeostasis. We find that licensing, in combination with differentiation occurring in pairs, is sufficient to maintain homeostasis and prevent stem cell extinction from stochastic fluctuations. Experimental data have shown that stem cells are competing for niche access, and our mathematical models demonstrate that this contributes to the reduction in the variability of stem cell numbers but does not prevent extinction. Hence, a combination of both regulation strategies, licensing with pairwise differentiation and competition for niche access, may be needed to reduce variability and prevent extinction simultaneously., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Introduction: Interstitial cells are crucial to the development of kidney structure and function, although the mechanism underlying their role in it remains unclear to date. Our previous study identified cell clusters in human fetal kidney tissue, and we further analyzed the interstitial cell cluster within this context., Methods: We extracted the barcoded cDNA from tissue samples and prepared spatial transcriptome libraries. Sequencing data were quality-checked, normalized, and clusters were identified using Seurat. Single-cell and spatial data were integrated using multimodal intersection analysis, and cell types were deconvoluted. DEGs in interstitial cells were identified and functionally annotated using DAVID. CellPhoneDB was used to predict ligand-receptor interactions between cell types., Results: The results of the present study revealed that this cluster of interstitial cells appeared to be scattered in the junction between the cortical and medullary regions. The subsequent Kyoto Encyclopedia of Genes and Genome pathway analysis revealed that the differentially expressed genes (DEGs) in this cluster of interstitial cells were involved in the WNT signaling pathway. The Gene Ontology (GO) analysis revealed that these DEGs were involved in multiple pathways associated with kidney development, with six of the genes (NKD2, TCF21, WNT5A, WNT4, MDK, and SFRP1) associated with kidney development exhibiting significant upregulation. Accordingly, it was inferred that these interstitial cells might be involved in regulating epithelial cell differentiation, ureteral bud development, and morphogenesis. The subsequent cell-cell communication analysis revealed that the cellular crosstalk was primarily regulated mainly by ligand-receptor pairs. Additionally, 17 genes reported to be associated with kidney disease were focused on, and these genes were found to be predominantly expressed in a single-cell type., Conclusion: In summary, the present study revealed the characteristics of a previously identified cluster of interstitial cells in the kidney tissue, thereby providing fresh insights into the process of kidney development., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

It has long been thought that the functional identity of mammalian brain neurons is programmed during development and remains stable throughout adult life; however, certain populations of neurons continue to express active regulators of neuronal identity into adulthood. Prolonged exposure to diet-induced metabolic stress induces features of neuronal identity modification in adult mice, and maladaptive changes in neuronal identity maintenance have been linked to cognitive impairment in humans suffering from neurodegenerative diseases often associated with obesity. Here we discuss how, by unraveling the neurological roots of obesity, we may solve the puzzle of whether mammalian brain neurons retain identity plasticity into adulthood, while advancing knowledge of the pathogenic mechanisms at the interface of metabolic and neurodegenerative disorders., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Glioblastomas (GBMs) are the most aggressive types of central nervous system tumors. Although certain genomic alterations have been identified as prognostic biomarkers of GBMs, the histomorphological features that predict their prognosis remain elusive. In this study, following an integrative diagnosis of 227 GBMs based on the 2021 World Health Organization classification system, the cases were histologically fractionated by cellular variations and abundance to evaluate the relationship between cellular heterogeneity and prognosis in combination with O-6-methylguanine-DNA methyltransferase gene promoter methylation (mMGMTp) status. GBMs comprised four major cell types: astrocytic, pleomorphic, gemistocytic, and rhabdoid cells. t-distributed stochastic neighbor embedding analysis using the histological abundance of heterogeneous cell types identified two distinct groups with significantly different prognoses. In individual cell component analysis, the abundance of gemistocytes showed a significantly favorable prognosis but confounding to mMGMTp status. Conversely, the abundance of epithelioid cells was correlated with the unfavorable prognosis. Linear model analysis showed the favorable prognostic utility of quantifying gemistocytic and epithelioid cells, independent of mMGMTp. The evaluation of GBM cell histomorphological heterogeneity is more effective for prognosis prediction in combination with mMGMTp analysis, indicating that histomorphological analysis is a practical and useful prognostication tool in an integrative diagnosis of GBMs., (© 2024. The Author(s).)

The incidence of central nervous system (CNS) disease has persistently increased over the last several years. There is an urgent need for effective methods to improve the cure rates of CNS disease. However, the precise molecular basis underlying the development and progression of major CNS diseases remains elusive. A complete molecular map will contribute to research on CNS disease treatment strategies. Emerging technologies such as single-cell RNA sequencing (scRNA-seq) and Spatial Transcriptomics (ST) are potent tools for exploring the molecular complexity, cell heterogeneity, and functional specificity of CNS disease. scRNA-seq and ST can provide insights into the disease at cellular and spatial transcription levels. This review presents a survey of scRNA-seq and ST studies on CNS diseases, such as chronic neurodegenerative diseases, acute CNS injuries, and others. These studies offer novel perspectives in treating and diagnosing CNS diseases by discovering new cell types or subtypes associated with the disease, proposing new pathophysiological mechanisms, uncovering novel therapeutic targets, and identifying putative biomarkers., (© 2024. The Author(s).)

Background: Neurodegenerative diseases are progressive disorders that severely diminish the quality of life of patients. However, research on neurodegenerative diseases needs to be refined and deepened. Single-cell polyomics is a technique for obtaining transcriptomic, proteomic, and other information from a single cell. In recent years, the heat of single-cell multiomics as an emerging research tool for brain science has gradually increased. Therefore, the aim of this study was to analyze the current status and trends of studies related to the application of single-cell multiomics in neurodegenerative diseases through bibliometrics., Result: A total of 596 publications were included in the bibliometric analysis. Between 2015 and 2022, the number of publications increased annually, with the total number of citations increasing significantly, exhibiting the fastest rate of growth between 2019 and 2022. The country/region collaboration map shows that the United States has the most publications and cumulative citations, and that China and the United States have the most collaborations. The institutions that produced the greatest number of articles were Harvard Medical School, Skupin, Alexander, and Wiendl. Among the authors, Heinz had the highest output. Mathys, H accumulated the most citations and was the authoritative author in the field. The journal Nature Communications has published the most literature in this field. A keyword analysis reveals that neurodegenerative diseases and lesions (e.g., Alzheimer's disease, amyloid beta) are the core and foundation of the field. Conversely, single-cell multiomics related research (e.g., single-cell RNA sequencing, bioinformatics) and brain nerve cells (e.g., microglia, astrocytes, neural stem cells) are the hot frontiers of this specialty. Among the references, the article "Single-cell transcriptomic analysis of Alzheimer's disease" is the most frequently cited (1,146 citations), and the article "Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq" was the most cited article in the field., Conclusion: The objective of this study is to employ bibliometric methods to visualize studies related to single-cell multiomics in neurodegenerative diseases. This will enable us to summarize the current state of research and to reveal key trends and emerging hotspots in the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Wang, Li, Liu, Wan and Liang.)

Fiber-reinforcement approaches have been used to replace aligned tissues with engineered constructs after injury or surgical resection, strengthening soft biomaterial scaffolds and replicating anisotropic, load-bearing properties. However, most studies focus on the macroscale aspects of these scaffolds, rarely considering the cell-biomaterial interactions that govern remodeling and extracellular matrix organization toward aligned neo-tissues. As initial cell-biomaterial responses within fiber-reinforced microenvironments likely influence the long-term efficacy of repair and regeneration strategies, here we elucidate the roles of spatial orientation, substrate stiffness, and matrix remodeling on early cell-fiber interactions. Bovine mesenchymal stromal cells (MSCs) were cultured in soft fibrin gels reinforced with a stiff 100 µm polyglycolide-co-caprolactone fiber. Gel stiffness and remodeling capacity were modulated by fibrinogen concentration and aprotinin treatment, respectively. MSCs were imaged at 3 days and evaluated for morphology, mechanoresponsiveness (nuclear Yes-associated protein [YAP] localization), and spatial features including distance and angle deviation from fiber. Within these constructs, morphological conformity decreased as a function of distance from fiber. However, these correlations were weak ( R 2 = 0.01043 for conformity and R 2 = 0.05542 for nuclear YAP localization), illustrating cellular heterogeneity within fiber-enforced microenvironments. To better assess cell-fiber interactions, we applied machine-learning strategies to our heterogeneous dataset of cell-shape and mechanoresponsive parameters. Principal component analysis (PCA) was used to project 23 input parameters (not including distance) onto 5 principal components (PCs), followed by agglomerative hierarchical clustering to classify cells into 3 groups. These clusters exhibited distinct levels of morpho-mechanoresponse (combination of morphological conformity and YAP signaling) and were classified as high response (HR), medium response (MR), and low response (LR) clusters. Cluster distribution varied spatially, with most cells (61%) closest to the fiber (0-75 µm) belonging to the HR cluster, and most cells (55%) furthest from the fiber (225-300 µm) belonging to the LR cluster. Modulation of gel stiffness and fibrin remodeling showed differential effects for HR cells, with stiffness influencing the level of mechanoresponse and remodeling capacity influencing the location of responding cells. Together, these novel findings demonstrate early trends in cellular patterning of the fiber-reinforced microenvironment, showing how spatial orientation, substrate biophysical properties, and matrix remodeling may guide the amplitude and localization of cellular mechanoresponses. These trends may guide approaches to optimize the design of microscale scaffold architecture and substrate properties for enhancing organized tissue assembly at the macroscale.

Background: Intratumor heterogeneity is common in cancers, with different cell subtypes supporting each other to become more malignant. Nasopharyngeal carcinoma (NPC), a highly metastatic cancer, shows significant heterogeneity among its cells. This study investigates how NPC cell subtypes with varying metastatic potentials influence each other through exosome-transmitted molecules., Methods: Exosomes were purified and characterized. MicroRNA expression was analyzed via sequencing and qRT-PCR. The effects of miR-30a-5p on migration, invasion, and metastasis were evaluated in vitro and in vivo. Its impact on desmoglein glycoprotein (DSG2) was assessed using dual-luciferase assays and Western blotting. Immunohistochemistry (IHC) and statistical models linked miR-30a-5p/DSG2 levels to patient prognosis., Results: Different NPC cell subtypes transmit metastatic potential via exosomes. High-metastatic cells enhance the migration, invasion, and metastasis of low-metastatic cells through exosome-transmitted miR-30a-5p. Plasma levels of exosomal miR-30a-5p are reliable indicators of NPC prognosis. miR-30a-5p may promote metastasis by targeting DSG2 and modulating Wnt signaling. Plasma exosomal miR-30a-5p inversely correlates with DSG2 levels, predicting patient outcomes., Conclusion: High-metastatic NPC cells can increase the metastatic potential of low-metastatic cells through exosome-transmitted miR-30a-5p, which is a valuable prognostic marker assessable via liquid biopsy., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the West China School of Medicine & West China Hospital of Sichuan University.)

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.

Biotic and abiotic environmental cues are major factors influencing plant growth and productivity. Interactions with biotic (e.g. symbionts and pathogens) and abiotic (e.g. changes in temperature, water, or nutrient availability) factors trigger signaling and downstream transcriptome adjustments in plants. While bulk RNA-sequencing technologies have traditionally been used to profile these transcriptional changes, tissue homogenization may mask heterogeneity of responses resulting from the cellular complexity of organs. Thus, whether different cell types respond equally to environmental fluctuations, or whether subsets of the responses are cell-type specific, are long-lasting questions in plant biology. The recent breakthrough of single-cell transcriptomics in plant research offers an unprecedented view of cellular responses under changing environmental conditions. In this review, we discuss the contribution of single-cell transcriptomics to the understanding of cell-type-specific plant responses to biotic and abiotic environmental interactions. Besides major biological findings, we present some technical challenges coupled to single-cell studies of plant-environment interactions, proposing possible solutions and exciting paths for future research., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Erythroid cells, the most prevalent cell type in blood, are one of the earliest products and permeate through the entire process of hematopoietic development in the human body, the oxygen-transporting function of which is crucial for maintaining overall health and life support. Previous investigations into erythrocyte differentiation and development have primarily focused on population-level analyses, lacking the single-cell perspective essential for comprehending the intricate pathways of erythroid maturation, differentiation, and the encompassing cellular heterogeneity. The continuous optimization of single-cell transcriptome sequencing technology, or single-cell RNA sequencing (scRNA-seq), provides a powerful tool for life sciences research, which has a particular superiority in the identification of unprecedented cell subgroups, the analyzing of cellular heterogeneity, and the transcriptomic characteristics of individual cells. Over the past decade, remarkable strides have been taken in the realm of single-cell RNA sequencing technology, profoundly enhancing our understanding of erythroid cells. In this review, we systematically summarize the recent developments in single-cell transcriptome sequencing technology and emphasize their substantial impact on the study of erythroid cells, highlighting their contributions, including the exploration of functional heterogeneity within erythroid populations, the identification of novel erythrocyte subgroups, the tracking of different erythroid lineages, and the unveiling of mechanisms governing erythroid fate decisions. These findings not only invigorate erythroid cell research but also offer new perspectives on the management of diseases related to erythroid cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rapid advancements in high-throughput single-cell RNA-seq (scRNA-seq) technologies and experimental protocols have led to the generation of vast amounts of transcriptomic data that populates several online databases and repositories. Here, we systematically examined large-scale scRNA-seq databases, categorizing them based on their scope and purpose such as general, tissue-specific databases, disease-specific databases, cancer-focused databases, and cell type-focused databases. Next, we discuss the technical and methodological challenges associated with curating large-scale scRNA-seq databases, along with current computational solutions. We argue that understanding scRNA-seq databases, including their limitations and assumptions, is crucial for effectively utilizing this data to make robust discoveries and identify novel biological insights. Such platforms can help bridge the gap between computational and wet lab scientists through user-friendly web-based interfaces needed for democratizing access to single-cell data. These platforms would facilitate interdisciplinary research, enabling researchers from various disciplines to collaborate effectively. This review underscores the importance of leveraging computational approaches to unravel the complexities of single-cell data and offers a promising direction for future research in the field., Competing Interests: AC is a co-founder of and serves as a Scientific Advisory Board member for LynxDx, Esanik Therapeutics, Medsyn, and Flamingo Therapeutics. AC is a scientific advisor or consultant for EdenRoc, Aurigene Oncology, Ascentage Pharma, Proteovant, Belharra, Rappta Therapeutics, and Tempus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gondal, Shah, Chinnaiyan and Cieslik.)

Breast cancer (BC) is currently the most common malignant tumour in women and one of the leading causes of their death around the world. New and increasingly personalised diagnostic and therapeutic tools have been introduced over the last few decades, along with significant advances regarding the study and knowledge related to BC. The tumour microenvironment (TME) refers to the tumour cell-associated cellular and molecular environment which can influence conditions affecting tumour development and progression. The TME is composed of immune cells, stromal cells, extracellular matrix (ECM) and signalling molecules secreted by these different cell types. Ever deeper understanding of TME composition changes during tumour development and progression will enable new and more innovative therapeutic strategies to become developed for targeting tumours during specific stages of its evolution. This review summarises the role of BC-related TME components and their influence on tumour progression and the development of resistance to therapy. In addition, an account on the modifications in BC-related TME components associated with therapy is given, and the completed or ongoing clinical trials related to this topic are presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cell types with different morphology, and function collaborate to maintain organ function. As such, analyzing proteomic differences and connections between different types of cells forms the foundation for establishing functional connectomes and developing in vitro organoid simulation experiments. However, the efficiency of cell type isolation from organs is limited by time, equipment, and cost. Here, hierarchical dendritic photonic crystal beads (HDPCBs) featuring high-density functional groups via the self-assembly of dendritic mesoporous structure SiO 2 nanoparticles (DM-SiO 2 ) and grafting dendrimers onto the surface of dendritic mesoporous photonic crystal beads (DMPCBs) is developed. This platform integrates multitype cell separation with in situ protein cleavage processes. Efficient simultaneous isolation of Kupffer cells and Liver Sinusoidal Endothelial cells (LSECs) from liver, with high specificity and convenient operation in a short separation time are demonstrated. The results reveal 2832 and 3442 unique proteins identified in Kupffer cells and LSECs using only 50 HDPCBs, respectively. 764 and 629 over-expressed proteins associated with the function of Kupffer cells and LSECs are found, respectively. The work offers a new method for efficiently isolating multiple cell types from tissues and downstream proteomic analysis, ultimately facilitating the identification of primary cell compositions and functions., (© 2024 Wiley‐VCH GmbH.)

Studying cell heterogeneity can provide a deeper understanding of biological activities, but appropriate studies cannot be performed using traditional bulk analysis methods. The development of diverse single cell bioanalysis methods is in urgent need and of great significance. Mass spectrometry (MS) has been recognized as a powerful technique for bioanalysis for its high sensitivity, wide applicability, label-free detection, and capability for quantitative analysis. In this review, the general development of single cell mass spectrometry (SCMS) field is covered. First, multiple existing SCMS techniques are described and compared. Next, the development of SCMS field is discussed in a chronological order. Last, the latest quantification studies on small molecules using SCMS have been described in detail., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Zhibo Yang reports financial support was provided by National Institutes of Health. Zhibo Yang reports financial support was provided by National Science Foundation. Zhibo Yang reports financial support was provided by The University of Oklahoma.

Rapid advancements in high-throughput single-cell RNA-seq (scRNA-seq) technologies and experimental protocols have led to the generation of vast amounts of genomic data that populates several online databases and repositories. Here, we systematically examined large-scale scRNA-seq databases, categorizing them based on their scope and purpose such as general, tissue-specific databases, disease-specific databases, cancer-focused databases, and cell type-focused databases. Next, we discuss the technical and methodological challenges associated with curating large-scale scRNA-seq databases, along with current computational solutions. We argue that understanding scRNA-seq databases, including their limitations and assumptions, is crucial for effectively utilizing this data to make robust discoveries and identify novel biological insights. Furthermore, we propose that bridging the gap between computational and wet lab scientists through user-friendly web-based platforms is needed for democratizing access to single-cell data. These platforms would facilitate interdisciplinary research, enabling researchers from various disciplines to collaborate effectively. This review underscores the importance of leveraging computational approaches to unravel the complexities of single-cell data and offers a promising direction for future research in the field., Competing Interests: 8Competing interests A.M.C. is a co-founder of and serves as a Scientific Advisory Board member for LynxDx, Esanik Therapeutics, Medsyn, and Flamingo Therapeutics. A.M.C. is a scientific advisor or consultant for EdenRoc, Aurigene Oncology, Ascentage Pharma, Proteovant, Belharra, Rappta Therapeutics, and Tempus.

Duck egg production, like that of laying hens, follows a typical low-peak-low cycle, reflecting the dynamics of the reproductive system. Post-peak, some ducks undergo a cessation of egg laying, indicative of a regression process in the oviduct. Notably, the magnum, being the longest segment of the oviduct, plays a crucial role in protein secretion. Despite its significance, few studies have investigated the molecular mechanisms underlying oviduct regression in ducks that have ceased laying eggs. In this study, we conducted single-cell transcriptome sequencing on the magnum tissue of Shaoxing ducks at 467 days of age, utilizing the 10× Genomics platform. This approach allowed us to generate a detailed magnum transcriptome map of both egg-laying and ceased-laying ducks. We collected transcriptome data from 13,708 individual cells, which were then subjected to computational analysis, resulting in the identification of 27 distinct cell clusters. Marker genes were subsequently employed to categorize these clusters into specific cell types. Our analysis revealed notable heterogeneity in magnum cells between the egg-laying and ceased-laying ducks, primarily characterized by variations in cells involved in protein secretion and extracellular matrix (ECM)-producing fibroblasts. Specifically, cells engaged in protein secretion were predominantly observed in the egg-laying ducks, indicative of their role in functional albumen deposition within the magnum, a phenomenon not observed in the ceased-laying ducks. Moreover, the proportion of THY1 + cells within the ECM-producing fibroblasts was found to be significantly higher in the egg-laying ducks (59%) compared to the ceased-laying ducks (24%). Similarly, TIMP4 + fibroblasts constituted a greater proportion of the ECM-producing fibroblasts in the egg-laying ducks (83%) compared to the ceased-laying ducks (58%). These findings suggest a potential correlation between the expression of THY1 and TIMP4 in ECM-producing fibroblasts and oviduct activity during functional reproduction. Our study provides valuable single-cell insights that warrant further investigation into the biological implications of fibroblast subsets in the degeneration of the reproductive tract. Moreover, these insights hold promise for enhancing the production efficiency of laying ducks.

As the largest organ of the body, the skin participates in various physiological activities, such as barrier function, sensory function, and temperature regulation, thereby maintaining the balance between the body and the natural environment. To date, compositional and transcriptional profiles in chicken skin cells have not been reported. Here, we report detailed transcriptome analyses of cell populations present in the skin of a black-feather chicken and a white-feather chicken using single-cell RNA sequencing (scRNA-seq). By analyzing cluster-specific gene expression profiles, we identified 12 cell clusters, and their corresponding cell types were also characterized. Subsequently, we characterized the subpopulations of keratinocytes, myocytes, mesenchymal cells, fibroblasts, and melanocytes. It is worth noting that we have identified a subpopulation of keratinocytes involved in pigment granule capture and a subpopulation of melanocytes involved in pigment granule deposition, both of which have a higher cell abundance in black-feather chicken compared to white-feather chicken. Meanwhile, we also compared the cellular heterogeneity features of Lueyang black-bone chicken skin with different feather colors. In addition, we also screened out 12 genes those could be potential markers of melanocytes. Finally, we validated the specific expression of SGK1, WNT5A, CTSC, TYR, and LAPTM5 in black-feather chicken, which may be the key candidate genes determining the feather color differentiation of Lueyang black-bone chicken. In summary, this study first revealed the transcriptome characteristics of chicken skin cells via scRNA-seq technology. These datasets provide valuable information for the study of avian skin characteristics and have important implications for future poultry breeding., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) hold immense promise for use in immunomodulation and regenerative medicine. However, their inherent heterogeneity makes it difficult to achieve optimal therapeutic outcomes for a specific clinical disease. Primed MSCs containing a certain cytokine can enhance their particular functions, thereby increasing their therapeutic potential for related diseases. Therefore, understanding the characteristic changes and underlying mechanisms of MSCs primed by various cytokines is highly important., Results: In this study, we aimed to reveal the cellular heterogeneity, functional subpopulations, and molecular mechanisms of MSCs primed with IFN-γ, TNF-α, IL-4, IL-6, IL-15, and IL-17 using single-cell RNA sequencing (scRNA-seq). Our results demonstrated that cytokine priming minimized the heterogeneity of the MSC transcriptome, while the expression of MSC surface markers exhibited only slight changes. Notably, compared to IL-6, IL-15, and IL-17; IFN-γ, TNF-α, and IL-4 priming, which stimulated a significantly greater number of differentially expressed genes (DEGs). Functional analysis, which included Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, indicated that IFN-γ, TNF-α, and IL-4-primed hUC-MSCs are involved in interferon-mediated immune-related processes, leukocyte migration, chemotaxis potential, and extracellular matrix and cell adhesion, respectively. Moreover, an investigation of various biological function scores demonstrated that IFN-γ-primed hUC-MSCs exhibit strong immunomodulatory ability, TNF-α-primed hUC-MSCs exhibit high chemotaxis potential, and IL-4-primed hUC-MSCs express elevated amounts of collagen. Finally, we observed that cytokine priming alters the distribution of functional subpopulations of MSCs, and these subpopulations exhibit various potential biological functions. Taken together, our study revealed the distinct regulatory effects of cytokine priming on MSC heterogeneity, biological function, and functional subpopulations at the single-cell level., Conclusions: These findings contribute to a comprehensive understanding of the inflammatory priming of MSCs, paving the way for their precise treatment in clinical applications., (© 2024. The Author(s).)

Chondrocyte is considered the only cell type in cartilage. However, the cell heterogeneity of chondrocytes in human articular cartilage is still not well defined, which hinders our understanding of the pathogenesis of osteoarthritis (OA). Here, we constructed a single-cell transcriptomic atlas of chondrocytes in healthy cartilage and identified nine chondrocyte subsets including homeostatic chondrocytes, proliferate fibrochondrocytes, and hypertrophic chondrocytes (HTC). Interestingly, we identified two distinct HTC subpopulations, among which HTC-1 specifically expressed genes associated with apoptosis and programmed cell death. We identified two main trajectories of chondrocytes, one of which differentiates into fibrochondrocytes, while the other terminates in apoptosis. Comparison of chondrocyte subsets between healthy and OA cartilage showed that proliferate fibrochondrocytes and HTC-1 expanded in OA patients, whereas homeostatic chondrocytes decreased. Interestingly, we discovered an OA-specific proliferate fibrochondrocyte subset that may contribute to the development of OA via inflammation. In summary, this study significantly enhanced our understanding of cell heterogeneity of chondrocytes in articular cartilage and provides insight into the pathogenesis of OA., (© 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltdé.)

Mesenchymal stem cells (MSCs) are adult stem cells that can be obtained, enriched and proliferated in vitro. They owned enormous potential in fields like regenerative medicine, tissue engineering and immunomodulation. However, though isolated from the same origin, MSCs are still essentially heterogeneous cell populations with different phenotypes and functions. This heterogeneity of MSCs significantly affects their therapeutic efficacy and brings obstacles to scientific research. Thus, reliable sorting technology which can isolate or purify MSC subpopulations with various potential and differentiation pathways is urgently needed. This review summarized principles, application status and clinical implications for these sorting methods, aiming at improving the understanding of MSC heterogeneity as well as providing fresh perspectives for subsequent clinical applications., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Marrow stimulation is a common reparative approach to treat injuries to cartilage and other soft tissues (e.g., rotator cuff). It involves the recruitment of bone marrow elements and mesenchymal stem cells (MSCs) into the defect, theoretically initiating a regenerative process. However, the resulting repair tissue is often weak and susceptible to deterioration with time. The populations of cells at the marrow stimulation site (beyond MSCs), and their contribution to inflammation, vascularity, and fibrosis, may play a role in quality of the repair tissue., Summary: In this review, we accomplish three goals: (1) systematically review clinical trials on the augmentation of marrow stimulation and evaluate their assumptions on the biological elements recruited; (2) detail the cellular populations in bone marrow and their impact on healing; and (3) highlight emerging technologies and approaches that could better guide these specific cell populations towards enhanced cartilage or soft tissue formation., Key Messages: We found that most clinical trials do not account for cell heterogeneity, nor do they specify the regenerative element recruited, and those that do typically utilize descriptions such as "clots," "elements," and "blood." Furthermore, our review of bone marrow cell populations demonstrates a dramatically heterogenous cell population, including hematopoietic cells, immune cells, fibroblasts, macrophages, and only a small population of MSCs. Finally, the field has developed numerous innovative techniques to enhance the chondrogenic potential (and reduce the anti-regenerative impacts) of these various cell types. We hope this review will guide approaches that account for cellular heterogeneity and improve marrow stimulation techniques to treat chondral defects., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To explore the molecular mechanisms of intestinal injury and treatment by analyzing changes in cellular heterogeneity and composition in rat ileal tissue during injury and treatment processes., Methods: We constructed a rat model of SAP and evaluated treatment with an injected of monoacylglycerol lipase (MAGL) inhibitor (JZL184) solution using three experimental groups: healthy male Sprague-Dawley (SD) rats injected with vehicle (CON), male SD SAP model rats injected with vehicle (SAP), and male SAP rats injected with JZL184. We obtained and prepared a single-cell suspension of ileal tissue of each rat for single-cell transcriptome sequencing., Results: This project classified changes in cellular heterogeneity and composition in rat ileal tissue during SAP-induced intestinal injury and MAGL treatment. We found that the number of fibroblast clusters was decreased in the SAP group relative to the CON group, and increased after JZL184 treatment. Further analysis of differences in gene expression between cell clusters in each group reveals that fibroblasts had the greatest number of differentially expressed genes. Most notably, expression of genes involved in communication between cells was found to vary during SAP-induced intestinal injury and JZL184 treatment. Among these changes, the degree of difference in expression of genes involved in communication between fibroblasts and other cells was the highest, indicating that fibroblasts in rat ileal tissue affect intestinal injury and repair through cell-to-cell communication. In addition, our results reveal that differentially expressed RNA-binding proteins in fibroblasts may affect their functions in intestinal injury and treatment by affecting the expression of genes regulating communication between cells., Conclusion: These findings emphasize the importance of understanding the interactions between fibroblasts and other cells in the context of intestinal injury, providing valuable insights for further exploring molecular mechanisms and insight for discovering new treatment targets and strategies., Competing Interests: The authors have confirmed that they have no potential conflicts of interest for this work., (© 2023 Wang et al.)

The present view on heartbeat initiation is that a primary pacemaker cell or a group of cells in the sinoatrial node (SAN) center paces the rest of the SAN and the atria. However, recent high-resolution imaging studies show a more complex paradigm of SAN function that emerges from heterogeneous signaling, mimicking brain cytoarchitecture and function. Here, we developed and tested a new conceptual numerical model of SAN organized similarly to brain networks featuring a modular structure with small-world topology. In our model, a lower rate module leads action potential (AP) firing in the basal state and during parasympathetic stimulation, whereas a higher rate module leads during β-adrenergic stimulation. Such a system reproduces the respective shift of the leading pacemaker site observed experimentally and a wide range of rate modulation and robust function while conserving energy. Since experimental studies found functional modules at different scales, from a few cells up to the highest scale of the superior and inferior SAN, the SAN appears to feature hierarchical modularity, i.e., within each module, there is a set of sub-modules, like in the brain, exhibiting greater robustness, adaptivity, and evolvability of network function. In this perspective, our model offers a new mainframe for interpreting new data on heterogeneous signaling in the SAN at different scales, providing new insights into cardiac pacemaker function and SAN-related cardiac arrhythmias in aging and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Maltsev, Stern, Lakatta and Maltsev.)

Our understanding of the pathophysiology of pulmonary arterial hypertension (PAH) has evolved over recent years, with the recognition that endothelial cell (EC) dysfunction and inflammation play an integral role in the development of this disease. ECs within the pulmonary vasculature play a unique role in maintaining vascular integrity and barrier function, regulating gas exchange, and contributing to vascular tone. Using single-cell transcriptomics, research has shown that there are multiple, unique EC subpopulations with different phenotypes. In response to injury or certain stressors such as hypoxia, there can be a dysregulated response with aberrant endothelial injury repair involving other pulmonary vascular cells and even immune cells. This aberrant signaling cascade is potentially a primary driver of pulmonary arterial remodeling in PAH. Recent studies have examined the role of EC clonal expansion, immune dysregulation, and genetic mutations in the pathogenesis of PAH. This review summarizes the existing literature on EC subpopulations and the intricate mechanisms through which ECs develop aberrant physiologic phenotypes and contribute to PAH. Our goal is to provide a framework for understanding the unique pulmonary EC biology and pathophysiology that is involved in the development of PAH., Competing Interests: The authors declare that this review article was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Newcomb and Farkas.)

Growth-restricted placentae have a reduced vascular network, impairing exchange of nutrients and oxygen. However, little is known about the differentiation events and cell types that underpin normal/abnormal placental vascular formation and function. Here, we used 23-colour flow cytometry to characterize placental vascular/perivascular populations between first trimester and term, and in foetal growth restriction (FGR). First-trimester endothelial cells had an immature phenotype (CD144+/lowCD36-CD146low), while term endothelial cells expressed mature endothelial markers (CD36+CD146+). At term, a distinct population of CD31low endothelial cells co-expressed mesenchymal markers (CD90, CD26), indicating a capacity for endothelial to mesenchymal transition (EndMT). In FGR, compared with normal pregnancies, endothelial cells constituted 3-fold fewer villous core cells (P < 0.05), contributing to an increased perivascular: endothelial cell ratio (2.6-fold, P < 0.05). This suggests that abnormal EndMT may play a role in FGR. First-trimester endothelial cells underwent EndMT in culture, losing endothelial (CD31, CD34, CD144) and gaining mesenchymal (CD90, CD26) marker expression. Together this highlights how differences in villous core cell heterogeneity and phenotype may contribute to FGR pathophysiology across gestation., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Single cell sequencing technology has provided unprecedented opportunities for comprehensively deciphering cell heterogeneity. Nevertheless, the high dimensionality and intricate nature of cell heterogeneity have presented substantial challenges to computational methods. Numerous novel clustering methods have been proposed to address this issue. However, none of these methods achieve the consistently better performance under different biological scenarios. In this study, we developed CAKE, a novel and scalable self-supervised clustering method, which consists of a contrastive learning model with a mixture neighborhood augmentation for cell representation learning, and a self-Knowledge Distiller model for the refinement of clustering results. These designs provide more condensed and cluster-friendly cell representations and improve the clustering performance in term of accuracy and robustness. Furthermore, in addition to accurately identifying the major type cells, CAKE could also find more biologically meaningful cell subgroups and rare cell types. The comprehensive experiments on real single-cell RNA sequencing datasets demonstrated the superiority of CAKE in visualization and clustering over other comparison methods, and indicated its extensive application in the field of cell heterogeneity analysis. Contact: Ruiqing Zheng. (rqzheng@csu.edu.cn)., (© The Author(s) 2023. Published by Oxford University Press.)

Single-cell RNA sequencing (scRNA-seq), which profiles gene expression at the cellular level, has effectively explored cell heterogeneity and reconstructed developmental trajectories. With the increasing research on diseases and biological processes, scRNA-seq datasets are accumulating rapidly, highlighting the urgent need for collecting and processing these data to support comprehensive and effective annotation and analysis. Here, we have developed a comprehensive S ingle- C ell transcriptome inte gration database fo r human and mouse (SCInter, https://bio.liclab.net/SCInter/index.php), which aims to provide a manually curated database that supports the provision of gene expression profiles across various cell types at the sample level. The current version of SCInter includes 115 integrated datasets and 1016 samples, covering nearly 150 tissues/cell lines. It contains 8016,646 cell markers in 457 identified cell types. SCInter enabled comprehensive analysis of cataloged single-cell data encompassing quality control (QC), clustering, cell markers, multi-method cell type automatic annotation, predicting cell differentiation trajectories and so on. At the same time, SCInter provided a user-friendly interface to query, browse, analyze and visualize each integrated dataset and single cell sample, along with comprehensive QC reports and processing results. It will facilitate the identification of cell type in different cell subpopulations and explore developmental trajectories, enhancing the study of cell heterogeneity in the fields of immunology and oncology., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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., (© 2023. The Author(s).)

If biomanufacturing can become a sustainable route for producing chemicals, it will provide a critical step in reducing greenhouse gas emissions to fight climate change. However, efforts to industrialize microbial synthesis of chemicals have met with varied success, due, in part, to challenges in translating laboratory successes to industrial scale. With a particular focus on Escherichia coli, this review examines the lessons learned when studying microbial physiology and metabolism under conditions that simulate large-scale bioreactors and methods to minimize cellular waste through reduction of maintenance energy, optimizing the stress response and minimizing culture heterogeneity. With general strategies to overcome these challenges, biomanufacturing process scale-up could be de-risked and the time and cost of bringing promising syntheses to market could be reduced., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Although distinct epithelial cell types have been distinguished in glandular tissues such as the mammary gland, the extent of heterogeneity within each cell type and the degree of endocrine control of this diversity across development are incompletely understood. By combining mass cytometry and cyclic immunofluorescence, we define a rich array of murine mammary epithelial cell subtypes associated with puberty, the estrous cycle, and sex. These subtypes are differentially proliferative and spatially segregate distinctly in adult versus pubescent glands. Further, we identify systematic suppression of lineage programs at the protein and RNA levels as a common feature of mammary epithelial expansion during puberty, the estrous cycle, and gestation and uncover a pervasive enrichment of ribosomal protein genes in luminal cells elicited specifically during progesterone-dominant expansionary periods. Collectively, these data expand our knowledge of murine mammary epithelial heterogeneity and connect endocrine-driven epithelial expansion with lineage suppression., Competing Interests: Declaration of interests J.S.B. is a member of the scientific advisory boards (SABs) of Frontier Medicines and eFFECTOR Therapeutics and was a member of the SAB of Agios Pharmaceuticals until January 2022. These roles do not constitute any conflicts of interest with this work. G.K.G. is of no relation to the Gray Foundation leadership., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint inhibitors (ICIs) are an expanding class of immunotherapeutic agents with the potential to cure cancer. Despite the outstanding clinical response in patient subsets, most individuals become refractory or develop resistance. Patient stratification and personalized immunotherapies are limited by the absence of predictive response markers. Recent findings show that dominant patterns of immune cell composition, T-cell status and heterogeneity, and spatiotemporal distribution of immune cells within the tumor microenvironment (TME) are becoming essential determinants of prognosis and therapeutic response. In this context, ICIs also function as investigational tools and proof of concept, allowing the validation of the identified mechanisms. After reviewing the current state of ICIs, this article will explore new comprehensive predictive markers for ICIs based on recent discoveries. We will discuss the recent establishment of a classification of TMEs into immune archetypes as a tool for personalized immune profiling, allowing patient stratification before ICI treatment. We will discuss the developing comprehension of T-cell diversity and its role in shaping the immune profile of patients. We describe the potential of strategies that score the mutual spatiotemporal modulation between T-cells and other cellular components of the TME. Additionally, we will provide an overview of a range of synthetic and naturally occurring or derived small molecules. We will compare compounds that were recently identified by in silico prediction to wet lab-validated drug candidates with the potential to function as ICIs and/or modulators of the cellular components of the TME., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the abundant infiltration of tumor microenvironment (TME) cells. The activity of TME cells reportedly plays an important role in the biology of FL. TME cells that reside within neoplastic follicles, such as T-follicular helper cells and follicular dendritic cells, have been shown to aid in FL development and progression through interactions with malignant B cells, whereas regulatory T cells have unexpectedly shown an apparently favorable prognostic impact in FL. Unfortunately, the understanding of the FL TME, particularly regarding minor cell subsets, has been hampered by unknown cell heterogeneity. As with other solid and hematologic cancers, novel single-cell analysis technologies have recently been applied to FL research and have uncovered previously unrecognized heterogeneities, not only in malignant B cells but also in TME cells. These reports have greatly increased the resolution of our understanding of the FL TME and, at the same time, raised questions about newly identified TME cells. This review provides an overview of the unique aspects of FL TME cells with a clinical viewpoint and highlights recent discoveries from single-cell analysis, while also suggesting potential future directions.

The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine.

Temperature is one of the main environmental cues affecting plant growth and development, and plants have evolved multiple mechanisms to sense and acclimate to high temperature. Emerging research has shown that transcription factors, epigenetic factors, and their coordination are essential for plant temperature responses and the resulting phenological adaptation. Here, we summarize recent advances in molecular and cellular mechanisms to understand how plants acclimate to high temperature and describe how plant meristems sense and integrate environmental signals. Furthermore, we lay out future directions for new technologies to reveal heterogeneous responses in different cell types thus improving plant environmental plasticity., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023. Published by Elsevier Ltd.)

In cancer, cell migration contributes to the spread of tumor cells resulting in metastasis. Heterogeneity in the migration capacity can produce individual cells with heightened capacity leading to invasion and metastasis. Our hypothesis is that cell migration characteristics can divide asymmetrically in mitosis, allowing a subset of cells to have a larger contribution to invasion and metastasis. Therefore, our aim is to elucidate whether sister cells have different migratory capacity and analyze if this difference is defined by mitosis. Through time-lapse videos, we analyzed migration speed, directionality, maximum displacement of each trajectory, and velocity as well as cell area and polarity and then compared the values between mother-daughter cells and between sister cells of three tumor cell lines (A172, MCF7, SCC25) and two normal cell lines (MRC5 and CHO·K1 cells). We observed that daughter cells had a different migratory phenotype compared to their mothers, and one single mitosis is enough for the sisters behave like nonrelated cells. However, mitosis did not influence cell area and polarity dynamics. These findings indicates that migration performance is not heritable, and that asymmetric cell division might have an important impact on cancer invasion and metastasis, by producing cells with different migratory capacity., Competing Interests: Declaration of competing interest None, (Copyright © 2023. Published by Elsevier Inc.)

Nine major cell populations among 46,716 cells were identified in mouse intestinal ischemia‒reperfusion (II/R) injury by single-cell RNA sequencing. For enterocyte cells, 11 subclusters were found, in which enterocyte cluster 1 (EC1), enterocyte cluster 3 (EC3), and enterocyte cluster 8 (EC8) were newly discovered cells in ischemia 45 min/reperfusion 720 min (I 45 min/R 720 min) group. EC1 and EC3 played roles in digestion and absorption, and EC8 played a role in cell junctions. For TA cells, after ischemia 45 min/reperfusion 90 min (I 45 min/R 90 min), many TA cells at the stage of proliferation were identified. For Paneth cells, Paneth cluster 3 was observed in the resting state of normal jejunum. After I 45 min/R 90 min, three new subsets were found, in which Paneth cluster 1 had good antigen presentation activity. The main functions of goblet cells were to synthesize and secrete mucus, and a novel subcluster (goblet cluster 5) with highly proliferative ability was discovered in I 45 min/R 90 min group. As a major part of immune system, the changes in T cells with important roles were clarified. Notably, enterocyte cells secreted Guca2b to interact with Gucy2c receptor on the membranes of stem cells, TA cells, Paneth cells, and goblet cells to elicit intercellular communication. One marker known as glutathione S-transferase mu 3 (GSTM3) affected intestinal mucosal barrier function by adjusting mitogen-activated protein kinases (MAPK) signaling during II/R injury. The data on the heterogeneity of intestinal cells, cellular communication and the mechanism of GSTM3 provide a cellular basis for treating II/R injury., Competing Interests: The authors declare that there are no conflicts of interest., (© 2023 The Author(s).)

The mechanisms that specify and stabilize cell subtypes remain poorly understood. Here, we identify two major subtypes of pancreatic β cells based on histone mark heterogeneity (β HI and β LO ). β HI cells exhibit ∼4-fold higher levels of H3K27me3, distinct chromatin organization and compaction, and a specific transcriptional pattern. β HI and β LO cells also differ in size, morphology, cytosolic and nuclear ultrastructure, epigenomes, cell surface marker expression, and function, and can be FACS separated into CD24 + and CD24 - fractions. Functionally, β HI cells have increased mitochondrial mass, activity, and insulin secretion in vivo and ex vivo. Partial loss of function indicates that H3K27me3 dosage regulates β HI /β LO ratio in vivo, suggesting that control of β cell subtype identity and ratio is at least partially uncoupled. Both subtypes are conserved in humans, with β HI cells enriched in humans with type 2 diabetes. Thus, epigenetic dosage is a novel regulator of cell subtype specification and identifies two functionally distinct β cell subtypes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging. Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo . Results : Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level. Discussion : These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spratt, Mizuguchi, Akaboshi, Kosakamoto, Okada, Obata and Ozeki.)

Introduction: Innate lymphoid cells (ILCs) are key components of the immune system, yet the similarity and distinction of the properties across tissues under homeostasis, inflammation and tumor process remain elusive., Methods: Here we performed integrative inference of ILCs to reveal their transcriptional profiles and heterogeneity from single-cell genomics. We collected a large number of ILCs from human six different tissues which can represent unique immune niches (circulation, lymphoid tissue, normal and inflamed mucosa, tumor microenvironment), to systematically address the transcriptional imprinting., Results: ILCs are profoundly imprinted by their organ of residence, and tissue-specific distinctions are apparent under pathological conditions. In the hepatocellular carcinoma microenvironment, we identified intermediate c-kit + ILC2 population, and lin - CD127 - NK-like cells that expressed markers of cytotoxicity including CCL5 and IFNG . Additionally, CD127 + CD94 + ILC1s were preferentially enriched in inflamed ileum from patients with Crohn's disease., Discussion: These analyses depicted a comprehensive characterization of ILC anatomical distribution and subset heterogeneity, and provided a base line for future temporal or spatial studies focused on tissue-specific ILC-mediated immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Song, Cao, Mao, Ai, Sun, Hu, Liu, Wang, Lu, Guan and Shen.)

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Silvano, Virgolini, Correia, Clarke, Isidro, Alves and Roldão.)

Satellite glial cells (SGCs) play an important role in regulating the function of trigeminal ganglion (TG) neurons. Multiple mediators are involved in the bidirectional communication between SGCs and neurons in different physiological and pathological states. However, molecular insights into the transcript characteristics of SGCs are limited. Moreover, little is known about the heterogeneity of SGCs in TG, and a more in-depth understanding of the interactions between SGCs and neuron subtypes is needed. Here we show the single-cell RNA sequencing (scRNA-seq) profile of SGCs in TG under physiological conditions. Our results demonstrate TG includes nine types of cell clusters, such as neurons, SGCs, myeloid Schwann cells (mSCs), non-myeloid Schwann cells (nmSCs), immune cells, etc., and the corresponding markers are also presented. We reveal the signature gene expression of SGCs, mSCs and nmSCs in the TG, and analyze the ligand-receptor pairs between neuron subtypes and SGCs in the TG. In the heterogeneity analysis of SGCs, four SGCs subtypes are identified, including subtypes enriched for genes associated with extracellular matrix organization, immediate early genes, interferon beta, and cell adhesion molecules, respectively. Our data suggest the molecular characteristics, heterogeneity of SGCs, and bidirectional interactions between SGCs and neurons, providing a valuable resource for studying SGCs in the TG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chu, Jia, Xu, Liu, Mai, Liu, Fan and Huang.)

Oligodendrocyte progenitor cells (OPCs) originate in the ventricular zone (VZ) of the brain and spinal cord, and their primary function is to differentiate into oligodendrocytes (OLs). Studies have shown that OPCs and OLs are pathologically and physiologically heterogeneous. Previous transcriptome analyses used Bulk RNA-seq, which compares average gene expression in cells and does not allow for heterogeneity. In recent years, the development of single-cell sequencing (scRNA-seq) and single-cell nuclear sequencing (snRNA-seq) has allowed us to study an individual cell. In this review, sc/snRNA-seq was used to study the different subpopulations of OL lineage cells, their developmental trajectories, and their applications in related diseases. These techniques can distinguish different subpopulations of cells, and identify differentially expressed genes in particular cell types under certain conditions, such as treatment or disease. It is of great significance to the study of the occurrence, prevention, and treatment of various diseases., (© 2022 International Society for Neurochemistry.)

Single-cell analysis of cell phenotypic information such as surface protein expression and nucleic acid content is essential for understanding heterogeneity within cell populations. Here the design and use of a dielectrophoresis-assisted self-digitization (SD) microfluidics chip is described; it captures single cells in isolated microchambers with high efficiency for single-cell analysis. The self-digitization chip spontaneously partitions aqueous solution into microchambers through a combination of fluidic forces, interfacial tension, and channel geometry. Single cells are guided to and trapped at the entrances of microchambers by dielectrophoresis (DEP) due to local electric field maxima created by an externally applied AC voltage. Excess cells are flushed away, and trapped cells are released into the chambers and prepared for in situ analysis by turning off the external voltage, by running reaction buffer through the chip, and by sealing the chambers with a flow of an immiscible oil phase through the surrounding channels. The use of this device in single-cell analysis is demonstrated by performing single-cell nucleic acid quantitation based on loop-mediated isothermal amplification (LAMP). This platform provides a powerful new tool for single-cell research pertaining to drug discovery. For example, the single-cell genotyping of cancer-related mutant gene observed from the digital chip could be useful biomarker for targeted therapy., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)