Transcriptome Profiling Research Articles

Microbial and Transcriptomic Landscape Associated With Neutrophil Extracellular Traps in Perianal Fistulizing Crohn's Disease.

Perianal fistulizing Crohn's disease (pfCD) poses significant healing challenges, closely associated with neutrophil extracellular traps (NETs). This study aimed to investigate the microbe-host interactions influencing NETs in pfCD. From January 2019 to July 2022, patients with pfCD were screened at Ren Ji Hospital. Patients in remission following comprehensive treatment were recruited. We documented clinical characteristics, medication regimens, healing outcomes, and infliximab levels in fistula tissues. NET positivity was confirmed by positive results in citrullinated histone H3 (CitH3) enzyme-linked immunosorbent assay (ELISA) and dual immunofluorescence staining for myeloperoxidase and CitH3. Microbial and transcriptomic profiles from fistula tissues, obtained during surgery, were analyzed using 16S rRNA gene sequencing and RNA sequencing. Differences in microbiome and transcriptomic profiles were evaluated, and their relationships were assessed using Mantel's and Spearman's coefficients. Significant differences in microbial communities were found between groups (P = .007). Representatively differential microbes such as Prevotella bivia, Streptococcus gordonii, and Bacteroides dorei were enriched in NETs-positive fistulas (P < .05). Functional analysis of microbes revealed reduced ubiquinol biosynthesis and butanoate production in NETs-negative fistulas (P < .05). Transcriptomic analysis indicated increased neutrophil and monocyte infiltration in NETs-positive fistulas, associated with pathways involving bacterial response, neutrophil chemotaxis, secretory processes, and peptidase activity (P < .05). Species prevalent in NETs-positive fistulas correlated positively with immune responses and wound healing pathways, whereas bacteria in NETs-negative fistulas correlated negatively. NETs were negatively associated with tissue infliximab levels (P = .001) and healing outcomes (P = .025). Our findings reveal unique microbial and transcriptomic signatures associated with NETs in pfCD, highlighting their profound influence on clinical outcomes.

AI-driven transcriptome profile-guided hit molecule generation

Denovo generation of bioactive and drug-like hit molecules is a pivotal goal in computer-aided drug discovery. While artificial intelligence (AI) has proven adept at generating molecules with desired chemical properties, previous studies often overlook the influence of disease-specific cellular environments. This study introduces GxVAEs, a novel AI-driven deep generative model designed to produce hit molecules from transcriptome profiles using dual variational autoencoders (VAEs). The first VAE, ProfileVAE, extracts latent features from transcriptome profiles to guide the second VAE, MolVAE, in generating hit molecules. GxVAEs aim to bridge the gap between molecule generation and the biological context of disease, producing molecules that are biologically relevant within specific cellular environments or pathological conditions. Experimental results and case studies focused on hit molecule generation demonstrate that GxVAEs surpass current state-of-the-art methods, in terms of reproducibility of known ligands. This approach is expected to effectively find potential molecular structures with bioactivities across diverse disease contexts.

Cryopreservation, cryoprotectants, and potential risk of epigenetic alteration.

The cryopreservation of gametes and embryos has increased notably over the past 20years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservationprocess is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming-crucial for the fate of the embryo-occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health.

Metabolite profiling and transcriptome analyses reveal defense regulatory network against pink tea mite invasion in tea plant

BackgroundThe tea plant Camellia sinensis (L.) O. Kuntze is a perennial crop, invaded by diversity of insect pest species, and pink tea mite is one of the most devastating pests for sustainable tea production. However, molecular mechanism of defense responses against pink tea mites in tea is still unknown. In this study, metabolomics and transcriptome profiles of susceptible and resistant tea varieties were compared before and after pink tea mite infestation.ResultsMetabolomics analysis revealed that abundance levels of polyphenol-related compounds changed significantly before and after infestation. At the transcript level, nearly 8 GB of clean reads were obtained from each sequenced library, and a comparison of infested plants of resistant and susceptible tea varieties revealed 9402 genes with significant differential expression. An array of genes enriched in plant pathogen interaction and biosynthetic pathways of phenylpropanoids showed significant differential regulation in response to pink tea mite invasion. In particular, the functional network linkage of disease resistant proteins, phenylalanine ammonia lyase, flavanone -3-hydroxylase, hydroxycinnamoyl-CoA shikimate transferase, brassinosteroid-6-oxidase 1, and gibberellin 2 beta-dioxygenase induced dynamic defense signals to suppress prolonged pink tea mite attacks. Further integrated analyses identified a complex network of transcripts and metabolites interlinked with precursors of various flavonoids that are likely modulate resistance against to pink tea mite.ConclusionsOur results characterized the profiles of insect induced metabolic and transcript reprogramming and identified a defense regulatory network that can potentially be used to fend off pink tea mites damage.

Stratification according to autoantibody status in systemic sclerosis reveals distinct molecular signatures

ObjectivesSystemic sclerosis (SSc) is a heterogeneous disease, complicating its management. Its complexity and the insufficiency of clinical manifestations alone to delineate homogeneous patient groups further challenge this task. However, autoantibodies...

Distinct Cutibacterium acnes subspecies defendens strains classified by multi-omics dissection alleviate inflammatory skin lesions of a rosacea-like mouse model

IntroductionCutibacterium acnes (C. acnes) resides in various organs such as the skin, prostate, eye, nose, stomach, and intestine, indicating the possibility of extensive crosstalk between this bacterium and the human body. C. acnes strains are classified into three subspecies based on phylogenetics and distinguishable phenotypes. Among them, C. acnes subsp. defendens strains are characterized by anti-inflammatory features, raising expectations for their potential as future microbiome therapeutics. However, the heterogeneity of C. acnes subsp. defendens and its corresponding immunological functions have not been clearly addressed.MethodsThe genetic diversity of the strains was assessed using single- and multi-locus sequence typing. Their immune-modulatory functions were evaluated in vitro using 2D and 3D assays with immune and epithelial cells. The anti-inflammatory effects were further confirmed in vivo using a rosacea-like mouse model. Comparative genomic and transcriptomic analyses were conducted to uncover mechanisms underlying the immunosuppressive activity of the strains.ResultsWe demonstrated that the newly isolated C. acnes subsp. defendens strains, exhibiting phenotypic heterogeneity, are distinctly clustered using single- and multi-locus sequence typing methods. These strains showed strong immune-regulatory functions in immune and epithelial cell-based 2D and 3D in vitro assays. Furthermore, their anti-inflammatory role was functionally confirmed in vivo using a rosacea-like mouse model, where they alleviated skin lesions characterized by hyperplasia and dermal inflammation. Comparative transcriptomics revealed that these strains may exert their immunosuppressive effects through the enhanced expression of acnecins and transcriptional variation in envelope stress regulators (specifically the two-component systems, CesSR homologs). Additionally, we propose that these C. acnes type II strains produce anti-inflammatory metabolites or peptides smaller than 3 kDa, which are associated with elevated pyrimidine and reduced L-arginine biosynthesis.DiscussionThe newly isolated C. acnes subsp. defendens strains demonstrate significant anti-inflammatory properties both in vitro and in vivo, suggesting their potential as microbiome-based therapeutics. Their unique genomic and transcriptomic profiles, including the production of small bioactive compounds and specific transcriptomic patterns, underpin their immunosuppressive capabilities. These findings provide a foundation for developing novel treatments for inflammatory skin conditions, such as rosacea.

Pathological autoantibody internalisation in myositis

ObjectivesAutoantibodies targeting intracellular proteins are common in various autoimmune diseases. In the context of myositis, the pathologic significance of these autoantibodies has been questioned due to the assumption that autoantibodies...

Expression-Driven Genetic Dependency Reveals Targets for Precision Medicine.

Cancer cells are heterogeneous, each harboring distinct molecular aberrations and are dependent on different genes for their survival and proliferation. While successful targeted therapies have been developed based on driver DNA mutations, many patient tumors lack druggable mutations and have limited treatment options. Here, we hypothesize that new precision oncology targets may be identified through "expression-driven dependency", whereby cancer cells with high expression of a targeted gene are more vulnerable to the knockout of that gene. We introduce a Bayesian approach, BEACON, to identify such targets by jointly analyzing global transcriptomic and proteomic profiles with genetic dependency data of cancer cell lines across 17 tissue lineages. BEACON identifies known druggable genes, e.g., BCL2, ERBB2, EGFR, ESR1, MYC , while revealing new targets confirmed by both mRNA- and protein-expression driven dependency. Notably, the identified genes show an overall 3.8-fold enrichment for approved drug targets and enrich for druggable oncology targets by 7 to 10-fold. We experimentally validate that the depletion of GRHL2 , TP63 , and PAX5 effectively reduce tumor cell growth and survival in their dependent cells. Overall, we present the catalog of express-driven dependency targets as a resource for identifying novel therapeutic targets in precision oncology.

Testing Neuroprotective Strategies in Prolonged Field Care Model of Traumatic Brain Injury and Hemorrhagic Shock.

Prolonged Field Care (PFC) is a military adaptation of Tactical Combat Casualty Care providing extended pre-hospital management during delayed extrication. Effects of addition of Valproic Acid (VPA) to Fresh Frozen Plasma (FFP) in a PFC model of hemorrhagic shock and traumatic brain injury (TBI) are not known. We hypothesized that VPA is associated with decreased neurological impairment, and its protective changes are detected at the transcriptomic level. Swine underwent TBI and 40%-blood volume hemorrhage. After 2-hours of shock, they were randomized to: 1)normal saline (NS), 2)NS+250 ml FFP (NS+FFP), or 3)NS+FFP+150 mg/kgVPA(NS+FFP+VPA). At 72-hours, they were transfused packed red blood cells before being euthanized. Intraoperative variables and neurological outcomes were compared. Brain lesion size was measured, and gene expression profiles were analyzed using RNA-sequencing. Pathway and network analyses were performed on differentially expressed genes. Real-time PCR was performed to validate key genes. NS+FFP and NS+FFP+VPA required significantly less crystalloid resuscitation(974 ml-NS+FFP; 1461ml-NS+FFP+VPA vs. 4540 ml-NS, p<0.001), had smaller brain lesion size(2477mm3-NS+FFP; 3018.0mm3-NS+FFP+VPA vs. 4517.0mm3-NS, p<0.01), and less functional neurologic impairment compared to NS. Per pathway analysis of differentially expressed genes, VPA was associated with enrichment of numerous metabolic changes in injured brains, which were not observed with FFP. Network analysis showed enrichment of various gene networks. MT-ATP8 gene was downregulated in VPA-treated animals. The addition of FFP to the resuscitation protocol resulted in a significant reduction in crystalloid requirements. Both, the FFP and FFP+VPA groups showed improved neurological recovery compared to NS alone and had distinctive transcriptomic profiles in injured brains at 72-hours. MT-ATP8, involved in worsening ischemia following brain injury, was down-regulated in VPA-treated animals.

Burkholderia pseudomallei BopE suppresses the Rab32-dependent defense pathway to promote its intracellular replication and virulence.

Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.IMPORTANCEB. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

Integrated transcriptomic analysis reveals dysregulated immune infiltration and pro-inflammatory cytokines in the secretory endometrium of recurrent implantation failure patients

Abstract Recurrent implantation failure (RIF) is a leading impediment to assisted reproductive technology, yet the underlying pathogenesis of RIF remains elusive. Recent studies have sought to uncover novel biomarkers and etiological factors of RIF by profiling transcriptomes of endometrial samples. Nonetheless, the inherent heterogeneity among published studies and a scarcity of experimental validations hinder the identification of robust markers of RIF. Hence, we integrated six publicly accessible datasets with 209 samples, including microarray profiles of endometrial samples in the secretory phase. After removing batch effects, we identified 175 differentially expressed genes. Gene set enrichment analysis identified dysregulation of immunological pathways in RIF. We also observed altered immune infiltration and pro-inflammatory cytokines in RIF. Protein–protein interaction network analysis identified ten hub genes, representing two co-expression modules significantly related to RIF. Knockdown of ENTPD3, one of the hub genes, promoted the epithelial-mesenchymal transition (EMT) process and resulted in elevated levels of pro-inflammatory cytokines. Collectively, our study reveals abnormal gene expressions involving the regulation of EMT and immune status in RIF, providing valuable insights into its pathogenesis.

Distinct molecular profiles and shared drug vulnerabilities in pancreatic metastases of renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common origin of pancreatic metastases (PM). Distinct genomic aberrations, favorable prognosis, and clinical observations on high angiogenesis, and succeeding tyrosine kinase inhibitor (TKI) sensitivity have been reported in PM-ccRCC. However, no functional or single-cell studies have been conducted thus far. We recruited five PM-ccRCC patients and investigated the genomic, single-cell transcriptomic, and drug sensitivity profiles of their patient-derived cells (PDCs). The PM depicted both expected and novel genomic alterations. Further, the transcriptomics differed from both primary and metastatic ccRCC, with upregulations of the PI3K/mTOR and - supporting the clinical observations - angiogenesis pathways. Data integration at pathway level showed that transcriptomics explained drug sensitivities the best. Accordingly, PM-ccRCC PDCs shared sensitivity to many PI3K/mTOR inhibitors. Altogether, we show distinct genomic and transcriptomic signatures in PM-ccRCC, highlight the superiority of transcriptomics in interpreting drug sensitivities, and encourage the use of TKIs and PI3K/mTOR inhibitors in PM-ccRCC.

Identification and analysis of the key genes for Escherichia coli heterologous protein expression by transcriptomic profiling.

Escherichia coli is a frequently used host for heterologous protein expression, but its expression efficiency is hindered by several limitations, such as formation of inclusion bodies and proteolytic degradation. In this study, we employed high-density fermentation of heterologous protein production in a 5-L bioreactor, resulting in a yield 2.25 times higher than that of the control group. Transcriptional analysis was conducted at three time points after induction for 0h, 4h, and 12h, revealing 420, 301, and 570 upregulated differentially expressed genes, as well as 424, 202, and 525 downregulated genes, respectively. By conducting enrichment analysis, we constructed strains that relieved without iron limitation, exhibiting a 36% increase in biomass and a 32% increase in protein expression. Furthermore, no overflow metabolism of acetic acid was detected during the protein expression process when utilizing chemostat culture, which indicated that the utilization efficiency of glucose was significantly enhanced without iron limitation. This study presents a novel approach to better comprehend the mechanism of high-yield production of heterologous proteins in Escherichia coli.

Similar humoral responses but distinct CD4+ T cell transcriptomic profiles in older adults elicited by MF59 adjuvanted and high dose influenza vaccines.

Older age (≥ 65 years) is associated with impaired responses to influenza vaccination, leading to the preferential recommendation of MF59-adjuvanted (MF59Flu) or high-dose (HDFlu) influenza vaccines for this age group in the United States. Herein, we characterized transcriptomic profiles of CD4+ T cells isolated from 234 recipients (≥ 65 years) of either MF59Flu or HDFlu vaccine, prior to vaccination and 28 days thereafter. We identified 412 and 645 differentially expressed genes (DEGs) in CD4+ T cells of older adults after receiving MF59Flu and HDFlu, respectively. DEGs in CD4+ T cells of MF59Flu recipients were enriched in 14 KEGG pathways, all of which were downregulated. DEGs in CD4+ T cells of HDFlu recipients were enriched in 11 upregulated pathways and 20 downregulated pathways. CD4+ T cells in both vaccine groups shared 50 upregulated genes and 75 downregulated genes, all of which were enriched in 7 KEGG pathways. The remaining 287 and 520 DEGs were specifically associated with MF59Flu and HDFlu, respectively. Unexpectedly, none of these DEGs was significantly correlated with influenza A/H3N2-specific HAI titers, suggesting these DEGs at the individual level may have a limited role in protection against influenza. Our findings emphasize the need for further investigation into other factors influencing immunity against influenza in older adults.

Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues.

Current methods for analyzing chromatin architecture are not readily scalable to heterogeneous tissues. Here we introduce Droplet Hi-C, which uses a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture of the mouse cortex and analyzed gene regulatory programs in major cortical cell types. In addition, we used this technique to detect copy number variations, structural variations and extrachromosomal DNA in human glioblastoma, colorectal and blood cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We refined the technique to allow joint profiling of chromatin architecture and transcriptome in single cells, facilitating exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C both addresses critical gaps in chromatin analysis of heterogeneous tissues and enhances understanding of gene regulation.

De novo transcriptome assembly and differential gene expression analysis in different developmental stages of Agriotes sputator (click beetle)

Wireworms, the larva of click beetle (Agriotes species), are one of the most destructive pests of horticultural crops in North America, responsible for considerable economic losses in Canada. Agriotes sputator (A. sputator) species is a predominant wireworm pest attacking potato fields in Eastern Canada. However, no information about its genome-wide gene expression profile, specifically for the genes involved with development is available to date. Therefore, we generated the transcriptome profile of A. sputator during five developmental stages, including the three larval stages and adult male and female click beetle. Out of 714.7 million raw reads, de novo assembly generated 564,561 transcripts. The data were subjected to differential expression analysis using DESeq2, gene ontology, annotation, and pathway analyses. A total of 34,709 differentially expressed genes (DEGs) were significant (log2 fold change > 2, padj < 0.05) across the developmental stages. Functional analysis of DEGs identified development signaling, metabolism, transport, cellular mechanisms, and drug metabolism (cytochrome p450) pathways. This study provides comprehensive sequence resources and potential gene differences at different developmental stages of A. sputator. These findings will represent a major step towards developing sustainable methods to control this widely distributed pest in agricultural fields.

Dynamic dysregulation of retrotransposons in neurodegenerative diseases at the single-cell level.

Retrotransposable elements (RTEs) are common mobile genetic elements comprising ∼42% of the human genome. RTEs play critical roles in gene regulation and function, but how they are specifically involved in complex diseases is largely unknown. Here, we investigate the cellular heterogeneity of RTEs using 12 single-cell transcriptome profiles covering three neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease, and multiple sclerosis. We identify cell type marker RTEs in neurons, astrocytes, oligodendrocytes, and oligodendrocyte precursor cells that are related to these diseases. The differential expression analysis reveals the landscape of dysregulated RTE expression, especially L1s, in excitatory neurons of multiple neurodegenerative diseases. Machine learning algorithms for predicting cell disease stage using a combination of RTE and gene expression features suggests dynamic regulation of RTEs in AD. Furthermore, we construct a single-cell atlas of retrotransposable elements in neurodegenerative disease (scARE) using these data sets and features. scARE has six feature analysis modules to explore RTE dynamics in a user-defined condition. To our knowledge, scARE represents the first systematic investigation of RTE dynamics at the single-cell level within the context of neurodegenerative diseases.

Description and functional validation of human enteroendocrine cell sensors.

Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including the incretins glucagon-like peptide 1 (GLP-1) and gastric inhibitory protein (GIP), which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and the existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in the secretion of hormones, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity, and mucosal immunity.

Single-Nuclei Transcriptome Profiling Reveals Intra-Tumoral Heterogeneity and Characterizes Tumor Microenvironment Architecture in a Murine Melanoma Model

Malignant melanoma is an aggressive cancer, with a high risk of metastasis and mortality rates, characterized by cancer cell heterogeneity and complex tumor microenvironment (TME). Single cell biology is an ideal and powerful tool to address these features at a molecular level. However, this approach requires enzymatic cell dissociation that can influence cellular coverage. By contrast, single nucleus RNA sequencing (snRNA-seq) has substantial advantages including compatibility with frozen samples and the elimination of a dissociation-induced, transcriptional stress response. To better profile and understand the functional diversity of different cellular components in melanoma progression, we performed snRNA-seq of 16,839 nuclei obtained from tumor samples along the growth of murine syngeneic melanoma model carrying a BRAFV600E mutation and collected 9 days or 23 days after subcutaneous cell injection. We defined 11 different subtypes of functional cell clusters among malignant cells and 5 different subsets of myeloid cells that display distinct global transcriptional program and different enrichment in early or advanced stage of tumor growth, confirming that this approach was useful to accurately identify intratumor heterogeneity and dynamics during tumor evolution. The current study offers a deep insight into the biology of melanoma highlighting TME reprogramming through tumor initiation and progression, underlying further discovery of new TME biomarkers which may be potentially druggable.

Unraveling the developmental heterogeneity within the human retina to reconstruct the continuity of retinal ganglion cell maturation and stage-specific intrinsic and extrinsic factors.

Tissue development is a complex spatiotemporal process with multiple interdependent components. Anatomical, histological, sequencing, and evolutional strategies can be used to profile and explain tissue development from different perspectives. The introduction of scRNAseq methods and the computational tools allows to deconvolute developmental heterogeneity and draw a decomposed uniform map. In this manuscript, we decomposed the development of a human retina with a focus on the retinal ganglion cells (RGC). To increase the temporal resolution of retinal cell classes maturation state we assumed the working hypothesis that that maturation of retinal ganglion cells is a continuous, non-discrete process. We have assembled the scRNAseq atlas of human fetal retina from fetal week 8 to week 27 and applied the computational methods to unravel maturation heterogeneity into a uniform maturation track. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified the continuous maturation track of RGC and described the cell-intrinsic (DEGs, maturation gene profiles, regulons, transcriptional motifs) and -extrinsic profiles (neurotrophic receptors across maturation, cell-cell interactions) of different RGC maturation states. We described the genes involved in the retina and RGC maturation, including de novo RGC maturation drivers. We demonstrate the application of the human fetal retina atlas as a reference tool, allowing automated annotation and universal embedding of scRNAseq data. Altogether, our findings deepen the current knowledge of the retina and RGC maturation by bringing in the maturation dimension for the cell class vs. state analysis. We show how the pseudotime application contributes to developmental-oriented analyses, allowing to order the cells by their maturation state. This approach not only improves the downstream computational analysis but also provides a true maturation track transcriptomics profile.