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Hepatitis B Virus-related acute-on-chronic liver failure (HBV-ACLF) is a severe complication with high fatality rates. However, the underlying mechanisms are still elusive and require further investigation. In this report, we described a case of type A HBV-ACLF in which significant changes were found in monocyte gene expression through single-cell RNA sequencing (scRNA-seq). Furthermore, we observed a shifted M1/M2 polarization as well as dynamic changes in HBV-ACLF markers expression within the circulating monocyte population. The co-expression of HBV-ACLF markers (MERTK, THBS1, PPARγ, and SEMA6B) in the circulating monocyte population suggests that monocytes could play an essential role in the development of HBV-ACLF. By analyzing a public HBV-ACLF cohort with bulk RNA-seq data (64 patients), we showed that the expression level of monocytes marker CD163 gradually increased among normal control individuals (NC, n = 15), patients with liver cirrhosis (LC, n = 10), patients with chronic hepatitis B infection (CHB, n = 10), patients with acute-on-chronic hepatic dysfunction (ACHD, n = 10), and patients with HBV-ACLF (n = 20). Furthermore, the representative HBV-ACLF marker THBS1 was significantly correlated with CD163 in this large clinical cohort. It indicated that the dramatic alteration in monocytes may not be limited to our type A HBV-ACLF patient alone but rather a common phenomenon in HBV-ACLF patients. Collectively, our scRNA-seq analysis showed that the pro-inflammatory status of monocytes had shifted into an anti-inflammatory status in this patient, indicating successful treatment and benign prognosis. Although scRNA-seq is still a time-consuming procedure and difficult to apply in daily clinical practice, this report preliminarily shows the promising potential utility of scRNA-seq in HBV-ACLF patients, by which altered status of monocytes could be unbiasedly detected.

Glioblastoma (GBM) cells have the potential to switch from being “proliferative cells” to peritumoral “invasive cells”. Peritumoral GBM cells have highly invasive properties that allow them to survive surgery, leading to recurrence. The mechanisms underlying the manner in which the tumor microenvironment (TME) regulates the invasiveness of GBM remain unclear. Single-cell RNA sequencing analysis revealed heterogeneity in GBM cells, microglia and macrophages. In this study, the Oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) expression indicated higher invasiveness in core GBM cells. Under environmental stress, the expression of OSMR and LIFR were up-regulated with the effect of hypoxic, acidic, and low-glucose conditions in vitro. Functional experiments revealed that TME stress significantly influences the proliferation, migration and invasion of GBM cells. The differences in core/peripheral TMEs in GBM affected the invasive properties, indicating the significant role of OSMR expression within the TME in tumor progression and postoperative therapy.

Background: Although PD-1 blockade is effective for treating several types of cancer, the efficacy of this agent in glioblastoma is largely limited. To overcome non-responders and the immunosuppressive tumor microenvironment, combinational immunotherapeutic strategies with anti-PD-1 need to be considered. Here, we developed IL-12-secreting mesenchymal stem cells (MSC_IL-12) with glioblastoma tropism and evaluated the therapeutic effects of anti-PD-1, MSC_IL-12, and their combination against glioblastoma. Methods: Therapeutic responses were evaluated using an immunocompetent mouse orthotopic model. Tumor-infiltrating lymphocytes (TILs) were analyzed using immunofluorescent imaging. Single-cell transcriptome was obtained from mouse brains after treatments. Results: Anti-PD-1 and MSC_IL-12 showed complete tumor remission in 25.0% (4/16) and 23.1% (3/13) of glioblastoma-implanted mice, respectively, and their combination yielded synergistic antitumor efficacy indicated by 50.0% (6/12) of complete tumor remission. Analyses of TILs revealed that anti-PD-1 increased CD8+ T cells, while MSC_IL-12 led to infiltration of CD4+ T cells and NK cells. Both therapies reduced frequencies of Tregs. All these aspects observed in each monotherapy group were superimposed in the combination group. Notably, no tumor growth was observed upon rechallenge in cured mice, indicating long-term immunity against glioblastoma provoked by the therapies. Single-cell RNA-seq data confirmed these results and revealed that the combined treatment led to immune-favorable tumor microenvironment–CD4+, CD8+ T cells, effector memory T cells, and activated microglia were increased, whereas exhausted T cells, Tregs, and M2 polarized microglia were reduced. Conclusion: Anti-PD-1 and MSC_IL-12 monotherapies show long-term therapeutic responses, and their combination further enhances antitumor efficacy against glioblastoma via inducing immune-favorable tumor microenvironment.

Colorectal cancer (CRC), also known as colon cancer, is the third most common cancer and the fourth most cause of cancer-related death in the world. CRC can be classified into two major subtypes, including microsatellite instability (MSI) and microsatellite stability (MSS), which showed different characteristics in immunotherapy. Low sensitivity of diagnostic biomarkers and metastasis are still the principal cause of mortality, especially in MSI. Here, applying computational programs, we identified recurring expression programs based on single cell RNA sequencing (scRNA-Seq) data of CRC cell lines. Notably, three MSI specific recurring modules were identified by non-negative matrix factorization (NMF). High NMF score genes enriched in the function of metabolism and inflammatory response. Focusing on top specific active transcription factor (TF), RUNX3 (Runt-related transcription factor 3), our results suggest that T cell infiltration was increased in RUNX3 high MSI CRC samples. Unbiased Gene Set Enrichment Analysis (GSEA) showed that RUNX3 was strongly associated with immune and metastasis related functions, such as Interferon Gamma (IFN-γ) and EPITHELIAL MESENCHYMAL TRANSITION (EMT). In addition, RUNX3 shows specific highly activated at epigenetic level in MSI compared with other gastrointestinal carcinomas. Positive correlation between RUNX3 and most immune checkpoints further confirmed RUNX3 might have crucial roles in MSI cancer progression and immunotherapy. Taken together, these results indicate significant tumor heterogeneity of two CRC subtypes at single-cell level and epigenetic modification level. These results also linked transcriptional dysregulation with immune infiltration at single-cell level in MSI, which may advance the application of scRNA-Seq technology in immunotherapy and contribute to developing novel biomarkers of this malignancy.

Background: Malignant tumours, particularly non-small cell lung cancer (NSCLC), pose a significant threat to human health due to their prevalence and lethality. Treatment methods for NSCLC vary greatly among individuals, making it crucial to identify predictive markers. Moreover, during tumour initiation and progression, tumour cells can release signaling molecules to induce polarization of macrophages towards a more tumour friendly M2 phenotype, which can promote tumour growth, metastasis, and drug resistance. Methods: We employed a comprehensive approach, combining bulk RNA-seq and single-cell sequencing analysis. Results: In our study, we used bulk RNA-seq and single-cell sequencing methods to analyze differential cells in NSCLC and adjacent tissues, searching for relevant marker genes that can predict prognosis and drug efficacy. We scrutinized biological phenomena such as macrophage-related gene methylation, copy number variation, and alternative splicing. Additionally, we utilized a co-culture technique of immune and tumour cells to explore the role of these genes in macrophage polarization. Our findings revealed distinct differences in macrophages between cancerous and adjacent tissues. We identified ANP32A, CCL20, ERAP2, MYD88, TMEM126B, TUBB6, and ZNF655 as macrophage-related genes that correlate with NSCLC patient prognosis and immunotherapy efficacy. Notably, ERAP2, TUBB6, CCL20, and TMEM126B can induce macrophage M0 to M2 polarization, promoting tumour proliferation. Conclusion: These findings significantly contribute to our understanding of the NSCLC tumour immune microenvironment. They pave the way for further research into the potential of these genes as targets for regulating tumour occurrence and development.

Nrf2, encoded by the gene Nfe2l2, is a broadly expressed transcription factor that regulates gene expression in response to reactive oxygen species (ROS) and oxidative stress. It is commonly referred to as a ubiquitous pathway, but this generalization overlooks work indicating that Nrf2 is essentially unexpressed in some neuronal populations. To explore whether this pattern extends throughout the central nervous system (CNS), we quantified Nfe2l2 expression and chromatin accessibility at the Nfe2l2 locus across multiple single cell datasets. In both the mouse and human CNS, Nfe2l2 was repressed in almost all mature neurons, but highly expressed in non-neuronal support cells, and this pattern was robust across multiple human CNS diseases. A subset of key Nrf2 target genes, like Slc7a11, also remained low in neurons. Thus, these data suggest that while most cells express Nfe2l2, with activity determined by ROS levels, neurons actively avoid Nrf2 activity by keeping Nfe2l2 expression low.

Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs. Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors. Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1. Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Introduction: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. Methods: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). Results: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. Conclusion: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.

Metastatic and locally advanced prostate cancer is treated by pharmacological targeting of androgen synthesis and androgen response via androgen signaling inhibitors (ASI), most of which target the androgen receptor (AR). However, ASI therapy invariably fails after 1–2 years. Emerging clinical evidence indicates that in response to ASI therapy, the AR-positive prostatic adenocarcinoma can transdifferentiate into AR-negative neuroendocrine prostate cancer (NEPC) in 17–25 % treated patients, likely through a process called neuroendocrine differentiation (NED). Despite high clinical incidence, the epigenetic pathways underlying NED and ASI therapy-induced NED remain unclear. By utilizing a combinatorial single cell and bulk mRNA sequencing workflow, we demonstrate in a time-resolved manner that following AR inhibition with enzalutamide, prostate cancer cells exhibit immediate loss of canonical AR signaling activity and simultaneous morphological change from epithelial to NE-like (NEL) morphology, followed by activation of specific neuroendocrine (NE)-associated transcriptional programs. Additionally, we observed that activation of NE-associated pathways occurs prior to complete repression of epithelial or canonical AR pathways, a phenomenon also observed clinically via heterogenous AR status in clinical samples. Our model indicates that, mechanistically, ASI therapy induces NED with initial morphological change followed by deactivation of canonical AR target genes and subsequent de-repression of NE-associated target genes, while retaining AR expression and transcriptional shift towards non-canonical AR activity. Coupled with scRNA-seq and CUT&RUN analysis, our model system can provide a platform for screening of potential therapeutic agents that may prevent ASI-induced NED or reverse the NED process.

The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells.

Objectives: Bacterial infection remains one of the greatest threats to human health. However, how human hosts respond to bacterial infection has not been thoroughly understood. Better understanding of this response will improve human health. Methods: Here, we conducted an investigation on host response to bacterial infection using unperturbed clinical samples and single-cell RNA-Seq (scRNA-Seq) technology. To evaluate immune alteration upon bacterial infection in scRNA-Seq data of peripheral blood mononuclear cells (PBMCs), we developed a barcode analytical framework named PBMCode. Results: Using this PBMCode framework, we revealed profound immune alteration in peripheral blood under bacterial infection, including the emergence of natural killer T (NKT) cell cluster, reduction of B cell population, and considerable changes in T cells and monocytes. In addition, we also observed a large quantity of low-density neutrophils. Conclusions: Our investigation on single cells provided unprecedented details in the alteration of both cell population and cell state under bacterial infection. These findings may be relevant to clinical decisions. The complexity of host response to bacterial infection revealed by scRNA-Seq deserves further attention in future studies.

The brain is a highly complex organ consisting of numerous types of cells with ample diversity at the epigenetic level to achieve distinct gene expression profiles. During neuronal cell specification, transcription factors (TFs) form regulatory modules with chromatin remodeling proteins to initiate the cascade of epigenetic changes. Currently, little is known about brain epigenetic regulatory modules and how they regulate gene expression in a cell-type specific manner. To infer TFs involved in neuronal specification, we applied a recursive motif search approach on the differentially methylated regions identified from single-cell methylomes. The epigenetic transcription regulatory modules (ETRM), including EGR1 and MEF2C, were predicted and the co-expression of TFs in ETRMs were examined with RNA-seq data from single or sorted brain cells using a conditional probability matrix. Lastly, computational predications were validated with EGR1 ChIP-seq data. In addition, methylome and RNA-seq data generated from Egr1 knockout mice supported the essential role of EGR1 in brain epigenome programming, in particular for excitatory neurons. In summary, we demonstrated that brain single cell methylome and RNA-seq data can be integrated to gain a better understanding of how ETRMs control cell specification. The analytical pipeline implemented in this study is freely accessible in the Github repository (https://github.com/Gavin-Yinld/brain_TF).

Background & Aims: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC., Methods: We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC., Results: We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC., Conclusions: Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis., Impact and Implications: This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The current study addresses the cellular complexity and plasticity of subcutaneous (inguinal) white adipose tissue (iWAT) in mice during the critical periods of perinatal growth and establishment. Methods: We performed a large-scale single cell transcriptomic (scRNA-seq) and epigenomic (snATAC-seq) characterization of cellular subtypes (adipose stromal cells (ASC) and adipocyte nuclei) during inguinal WAT (subcutaneous; iWAT) development in mice, capturing the early postnatal period (postnatal days (PND) 06 and 18) through adulthood (PND56). Results: Perinatal and adult iWAT contain 3 major ASC subtypes that can be independently identified by RNA expression profiles and DNA transposase accessibility. Furthermore, the transcriptomes and enhancer landscapes of both ASC and adipocytes dynamically change during postnatal development. Perinatal ASC (PND06) are highly enriched for several imprinted genes (IGs; e.g., Mest, H19, Igf2) and extracellular matrix proteins whose expression then declines prior to weaning (PND18). By comparison, adult ASC (PND56) are more enriched for transcripts associated with immunoregulation, oxidative stress, and integrin signaling. Two clusters of mature adipocytes, identified through single nuclei RNA sequencing (snRNA-seq), were distinctive for proinflammatory/immune or metabolic gene expression patterns that became more transcriptionally diverse in adult animals. Single nuclei assay for transposase-accessible chromatin (snATAC-seq) revealed that differences in gene expression were associated with developmental changes in chromatin accessibility and predicted transcription factor motifs (e.g., Plagl1, Ar) in both stromal cells and adipocytes. Conclusions: Our data provide new insights into transcriptional and epigenomic signaling networks important during iWAT establishment at a single cell resolution, with important implications for the field of metabolic programming.

Objective: Homo- or heterodimerization of G protein–coupled receptors (GPCRs) generally alters the normal functioning of these receptors and mediates their responses to a variety of physiological stimuli in vivo. It is well known that melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) are key regulators of appetite and energy homeostasis in the central nervous system (CNS). However, the GPCR partners of MC3R and MC4R are not well understood. Our objective is to analyze single cell RNA-seq datasets of the hypothalamus to explore and identify novel GPCR partners of MC3R and MC4R and examine the pharmacological effect on the downstream signal transduction and membrane translocation of melanocortin receptors. Methods: We conducted an integrative analysis of multiple single cell RNA-seq datasets to reveal the expression pattern and correlation of GPCR families in the mouse hypothalamus. The emerging GPCRs with important metabolic functions were selected for cloning and co-immunoprecipitation validation. The positive GPCR partners were then tested for the pharmacological activation, competitive binding assay and surface translocation ELISA experiments. Results: Based on the expression pattern of GPCRs and their function enrichment results, we narrowed down the range of potential GPCR interaction with MC3R and MC4R for further confirmation. Co-immunoprecipitation assay verified 23 and 32 novel GPCR partners that interacted with MC3R and MC4R in vitro. The presence of these GPCR partners exhibited different effects in the physiological regulation and signal transduction of MC3R and MC4R. Conclusions: This work represented the first large-scale screen for the functional GPCR complex of central melanocortin receptors and defined a composite metabolic regulatory GPCR network of the hypothalamic nucleuses.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons throughout the brain and spinal cord progressively degenerate resulting in muscle atrophy, paralysis and death. Recent studies using animal models of ALS implicate multiple cell-types (e.g., astrocytes and microglia) in ALS pathogenesis in the spinal motor systems. To ascertain cellular vulnerability and cell-type specific mechanisms of ALS in the brainstem that orchestrates oral-motor functions, we conducted parallel single cell RNA sequencing (scRNA-seq) analysis using the high-throughput Drop-seq method. We isolated 1894 and 3199 cells from the brainstem of wildtype and mutant SOD1 symptomatic mice respectively, at postnatal day 100. We recovered major known cell types and neuronal subpopulations, such as interneurons and motor neurons, and trigeminal ganglion (TG) peripheral sensory neurons, as well as, previously uncharacterized interneuron subtypes. We found that the majority of the cell types displayed transcriptomic alterations in ALS mice. Differentially expressed genes (DEGs) of individual cell populations revealed cell-type specific alterations in numerous pathways, including previously known ALS pathways such as inflammation (in microglia), stress response (ependymal and an uncharacterized cell population), neurogenesis (astrocytes, oligodendrocytes, neurons), synapse organization and transmission (microglia, oligodendrocyte precursor cells, and neuronal subtypes), and mitochondrial function (uncharacterized cell populations). Other cell-type specific processes altered in SOD1 mutant brainstem include those from motor neurons (axon regeneration, voltage-gated sodium and potassium channels underlying excitability, potassium ion transport), trigeminal sensory neurons (detection of temperature stimulus involved in sensory perception), and cellular response to toxic substances (uncharacterized cell populations). DEGs consistently altered across cell types (e.g., Malat1), as well as cell-type specific DEGs, were identified. Importantly, DEGs from various cell types overlapped with known ALS genes from the literature and with top hits from an existing human ALS genome-wide association study (GWAS), implicating the potential cell types in which the ALS genes function with ALS pathogenesis. Our molecular investigation at single cell resolution provides comprehensive insights into the cell types, genes and pathways altered in the brainstem in a widely used ALS mouse model.

Summary: Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.

Summary: To understand the cellular basis of behavior, it is necessary to know the cell types that exist in the nervous system and their contributions to function. Spinal networks are essential for sensory processing and motor behavior and provide a powerful system for identifying the cellular correlates of behavior. Here, we used massively parallel single nucleus RNA sequencing (snRNA-seq) to create an atlas of the adult mouse lumbar spinal cord. We identified and molecularly characterized 43 neuronal populations. Next, we leveraged the snRNA-seq approach to provide unbiased identification of neuronal populations that were active following a sensory and a motor behavior, using a transcriptional signature of neuronal activity. This approach can be used in the future to link single nucleus gene expression data with dynamic biological responses to behavior, injury, and disease.

Characterization of the cardiac cellulome, the network of cells that form the heart, is essential for understanding cardiac development and normal organ function and for formulating precise therapeutic strategies to combat heart disease. Recent studies have reshaped our understanding of cardiac cellular composition and highlighted important functional roles for non-myocyte cell types. In this study, we characterized single-cell transcriptional profiles of the murine non-myocyte cardiac cellular landscape using single-cell RNA sequencing (scRNA-seq). Detailed molecular analyses revealed the diversity of the cardiac cellulome and facilitated the development of techniques to isolate understudied cardiac cell populations, such as mural cells and glia. Our analyses also revealed extensive networks of intercellular communication and suggested prevalent sexual dimorphism in gene expression in the heart. This study offers insights into the structure and function of the mammalian cardiac cellulome and provides an important resource that will stimulate studies in cardiac cell biology.

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results. Whole blood samples from 20 healthy adults were collected and incubated at 20-25 °C for 2-48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2-4 h) and longer (8-48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs. PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased. PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities., Competing Interests: Declaration of competing interest All authors are employees and stockholders of Gilead Sciences Inc., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLC) have enormous potential as a replacement for primary hepatocytes in drug screening, toxicology and cell replacement therapy, but their genome-wide expression patterns differ strongly from primary human hepatocytes (PHH)., Methods: We differentiated human induced pluripotent stem cells (hiPSC) via definitive endoderm to HLC and characterized the cells by single-cell and bulk RNA-seq, with complementary epigenetic analyses. We then compared HLC to PHH and publicly available data on human fetal hepatocytes (FH) ex vivo; we performed bioinformatics-guided interventions to improve HLC differentiation via lentiviral transduction of the nuclear receptor FXR and agonist exposure., Results: Single-cell RNA-seq revealed that transcriptomes of individual HLC display a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH - mostly intestinal genes - within the same cell. Bulk-level overrepresentation analysis, as well as regulon analysis at the single-cell level, identified sets of regulatory factors discriminating HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and increased the ability of bile canalicular secretion as well as lipid droplet formation, thereby increasing HLCs' similarity to PHH. The undesired non-liver gene expression was reproducibly decreased, although only by a moderate degree., Conclusion: In contrast to physiological hepatocyte precursor cells and mature hepatocytes, HLC co-express liver and hybrid genes in the same cell. Targeted modification of the FXR gene regulatory network improves their differentiation by suppressing intestinal traits whilst inducing hepatocyte features., Lay Summary: Generation of human hepatocytes from stem cells represents an active research field but its success is hampered by the fact that the stem cell-derived 'hepatocytes' still show major differences to hepatocytes obtained from a liver. Here, we identified an important reason for the difference, specifically that the stem cell-derived 'hepatocyte' represents a hybrid cell with features of hepatocytes and intestinal cells. We show that a specific protein (FXR) suppresses intestinal and induces liver features, thus bringing the stem cell-derived cells closer to hepatocytes derived from human livers., Competing Interests: Conflict of interest Patricio Godoy is also affiliated with F. Hoffmann-La Roche Ltd (Roche Innovation Center Basel, Basel, Switzerland), Barbara Küppers-Munther is affiliated with Takara Bio Europe AB (former Cellartis AB) (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden). Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Previously, we have shown that Maternal Separation and Early Weaning (MSEW) exacerbates high fat diet (HF)-induced visceral obesity in female offspring compared to normally reared female mice. Stress hormones such as glucocorticoids and mineralocorticoids are critical mediators in the process of fat expansion, and both can activate the mineralocorticoid receptor (MR) in the adipocyte. Therefore, this study aimed to, comprehend the specific effects of MSEW on adipose tissue basic homeostatic function, and investigate whether female MSEW mice show an exacerbated obesogenic response mediated by MR. Gonadal white adipose tissue (gWAT), a type of visceral fat, was collected to assess lipidomics, transcriptomics, and in vitro lipolysis assay. Obese female MSEW mice showed increased adiposity, elevated 44:2/FA 18:2 + NH4 lipid class and reduced mitochondrial DNA density compared to obese control counterparts. In addition, single-cell RNA sequencing in isolated pre- and mature adipocytes showed a ~9-fold downregulation of aquaglycerolporin 3 (Aqp3), a channel responsible for glycerol efflux in adipocytes. Obese MSEW mice showed high levels of circulating aldosterone and gWAT-derived corticosterone compared to controls. Further, the MR blocker spironolactone (Spiro, 100 mg/kg/day, 2 weeks) normalized the elevated intracellular glycerol levels, the greater in vitro lipolysis response, and the number of large size adipocytes in MSEW mice compared to the controls. Our data suggests that MR plays a role promoting adipocyte hypertrophy in female MSEW mice by preventing lipolysis via glycerol release in favor of triglyceride formation and storage., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Myeloproliferative neoplasms (MPNs) is an umbrella term for several heterogenous diseases, which are characterized by their stem cell origin, clonal hematopoiesis and increase of blood cells of the myeloid lineage. The focus will be on BCR-ABL1 negative MPNs, polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET). Seminal findings in the field of MPN were driven by genomic analysis, focusing on dissecting genomic changes MPN patients. This led to identification of major MPN driver genes, JAK2, MPL and CALR. Transcriptomic analysis promises to bridge the gap between genetic and phenotypic characterization of each patient's tumor and with the advent of single cell sequencing even for each MPN cancer cell. This review will focus on efforts to mine the bulk transcriptome of MPN patients, including analysis of fusion genes and splicing alterations which can be addressed with RNA-seq technologies. Furthermore, this paper aims to review recent endeavors to elucidate tumor heterogeneity in MPN hematopoietic stem and progenitor cells using single cell technologies. Finally, it will highlight current shortcoming and future applications to advance the field in MPN biology and improve patient diagnostics using RNA-based assays., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time., Methods: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership)., Results: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies., Conclusion: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes., (© 2021 Published by Elsevier, Inc., on behalf of the International Society of Nephrology.)

Background: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs., Objectives: To characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis., Results: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state., Conclusions: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Objectives: Bacterial infection remains one of the greatest threats to human health. However, how human hosts respond to bacterial infection has not been thoroughly understood. Better understanding of this response will improve human health., Methods: Here, we conducted an investigation on host response to bacterial infection using unperturbed clinical samples and single-cell RNA-Seq (scRNA-Seq) technology. To evaluate immune alteration upon bacterial infection in scRNA-Seq data of peripheral blood mononuclear cells (PBMCs), we developed a barcode analytical framework named PBMCode., Results: Using this PBMCode framework, we revealed profound immune alteration in peripheral blood under bacterial infection, including the emergence of natural killer T (NKT) cell cluster, reduction of B cell population, and considerable changes in T cells and monocytes. In addition, we also observed a large quantity of low-density neutrophils., Conclusions: Our investigation on single cells provided unprecedented details in the alteration of both cell population and cell state under bacterial infection. These findings may be relevant to clinical decisions. The complexity of host response to bacterial infection revealed by scRNA-Seq deserves further attention in future studies., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Oocytes of better quality and developmental competence are highly demanded, which is affected by many intrinsic and external factors, including environmental pollutants. We have previously demonstrated that 7, 12-dimethylbenz [a]anthracene (DMBA) reduces the developmental competence of porcine oocytes, by desynchronizing nuclear and ooplasmic maturation. However, the underlying molecular mechanism remains obscure. Here we performed single cell RNA-seq to study the transcriptome changes in DMBA-treated porcine MII oocytes, and identified 19 protein-coding genes and 156 novel long non-coding RNAs (lncRNAs) with abundance to be significantly different (P < 0.05), which enriched in signaling pathways such as glycosphingolipid biosynthesis, nicotine addiction, basal transcription factors and nucleotide excision repair. RT-qPCR on oocyte pools confirmed ornithine aminotransferase (Oat) and serine/arginine-rich splicing factor 4 (Srsf4) to be significantly up- and down-regulated, respectively (P < 0.05). Treating porcine COCs with MAPK and PLC pathway inhibitors suppressed DMBA's effects on increasing PB1 extrusion rate. In addition, DMBA co-incubation with 250 μM vitamin C derivative (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) and 100 μM co-enzyme Q10 (CoQ10) could significantly reduce the DMBA-induced high ROS level, and partially alleviate the DMBA-induced high PB1 rate, whereas the cleavage and blastocyst rates of parthenotes derived from treated mature oocytes remained to be low. Collectively, our findings indicate that single cell RNA-seq can help reveal the dynamics of molecular signaling pathways for porcine oocytes treated by DMBA, and supplement of anti-oxidative reagents could not sufficiently rescue DMBA-induced defects of porcine oocytes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development., (Copyright © 2019. Published by Elsevier Inc.)

Directed differentiation of pluripotent stem cells provides an accessible system to model development. However, the distinct cell types that emerge, their dynamics, and their relationship to progenitors in the early embryo has been difficult to decipher because of the cellular heterogeneity inherent to differentiation. Here, we used a combination of bulk RNA-Seq, single cell RNA-Seq, and bioinformatics analyses to dissect the cell types that emerge during directed differentiation of mouse embryonic stem cells as embryoid bodies and we compared them to spatially and temporally resolved transcriptional profiles of early embryos. Our single cell analyses of the day 4 embryoid bodies revealed three populations which had retained related yet distinct pluripotent signatures that resemble the pre- or post-implantation epiblast, one population of presumptive neuroectoderm, one population of mesendoderm, and four populations of neural progenitors. By day 6, the neural progenitors predominated the embryoid bodies, but both a small population of pluripotent-like cells and an anterior mesoderm-like Brachyury-expressing population were present. By comparing the day 4 and day 6 populations, we identified candidate differentiation paths, transcription factors, and signaling pathways that mark the in vitro correlate of the transition from the mid-to-late primitive streak stage., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Gene expression heterogeneity contributes to development as well as disease progression. Due to technological limitations, most studies to date have focused on differences in mean expression across experimental conditions, rather than differences in gene expression variance. The advent of single cell RNA sequencing has now made it feasible to study gene expression heterogeneity and to characterise genes based on their coefficient of variation., Methods: We collected single cell gene expression profiles for 32 human and 39 mouse embryonic stem cells and studied correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further systematically characterised properties unique to High CV genes., Results: Highly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent (H3K4me3 and H3K27me3) marked promoters and showed enrichment for response to DNA damage and DNA repair., Conclusions: Taken together, this analysis demonstrates the divergent characteristics of genes based on their CV. High CV genes tend to form co-expression clusters and they explain bivalency at least in part., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)