Single-nucleus Transcriptomes Research Articles

Droplet-based transcriptome profiling of individual synapses.

Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model.

PS-B05-11: IMPAIRED CELL INTERACTIONS AT THE PRE-ECLAMPTIC MATERNAL-FOETAL INTERFACE

Hypertensive disorders in pregnancy, of which the multisystem syndrome pre-eclampsia is most severe, leading to preterm delivery, maternal mortality and life-long complications1. Pre-eclampsia lacks early screening tools2,3 and causal therapies4,5. To elucidate early disease dynamics, we present the first spatio-temporal study comparing single-nuclei transcriptomes of human preterm pre-eclamptic placentae and healthy controls, contextualising this in a comprehensive study including early and late gestational placentae and implementing spatial multi-omics methods. Here we show trophoblast pre-fusion cells and their developmental trajectory running towards a novel trophoblast cell-state we identify as juvenile syncytiotrophoblast. We found that disturbed trophoblast development, reconstructed by spatial multi-omics and including early dysregulation in differentiation-driving transcription factor p300, likely initiates pre-eclampsia. We suggest pre-eclampsia to be a disease of dysregulated syncytiotrophoblast differentiation: Disease-specific premature differentiation of the maternal-foetal barrier leads to a senescent syncytiotrophoblastic interaction through maternal circulation with maternal vessels. This initiates a disrupted decidual maternal response via LEP, GDF15 and INHBA. Our results highlight a disturbed cellular communication from foetal to maternal side during the development of pre-eclampsia starting with a disturbed trophoblast maturation. We provide new targets for potential early disease prevention in order to protect mother and child from increased gestational mortality and morbidity but also from life-long increased cardiovascular disease risk.

The gold-ringed octopus (Amphioctopus fangsiao) genome and cerebral single-nucleus transcriptomes provide insights into the evolution of karyotype and neural novelties

BackgroundColeoid cephalopods have distinctive neural and morphological characteristics compared to other invertebrates. Early studies reported massive genomic rearrangements occurred before the split of octopus and squid lineages (Proc Natl Acad Sci U S A 116:3030-5, 2019), which might be related to the neural innovations of their brain, yet the details remain elusive. Here we combine genomic and single-nucleus transcriptome analyses to investigate the octopod chromosome evolution and cerebral characteristics.ResultsWe present a chromosome-level genome assembly of a gold-ringed octopus, Amphioctopus fangsiao, and a single-nucleus transcriptome of its supra-esophageal brain. Chromosome-level synteny analyses estimate that the chromosomes of the ancestral octopods experienced multiple chromosome fission/fusion and loss/gain events by comparing with the nautilus genome as outgroup, and that a conserved genome organization was detected during the evolutionary process from the last common octopod ancestor to their descendants. Besides, protocadherin, GPCR, and C2H2 ZNF genes are thought to be highly related to the neural innovations in cephalopods (Nature 524:220–4, 2015), and the chromosome analyses pinpointed several collinear modes of these genes on the octopod chromosomes, such as the collinearity between PCDH and C2H2 ZNF, as well as between GPCR and C2H2 ZNF. Phylogenetic analyses show that the expansion of the octopod protocadherin genes is driven by a tandem-duplication mechanism on one single chromosome, including two separate expansions at 65 million years ago (Ma) and 8–14 Ma, respectively. Furthermore, we identify eight cell types (i.e., cholinergic and glutamatergic neurons) in the supra-esophageal brain of A. fangsiao, and the single-cell expression analyses reveal the co-expression of protocadherin and GPCR in specific neural cells, which may contribute to the neural development and signal transductions in the octopod brain.ConclusionsThe octopod genome analyses reveal the dynamic evolutionary history of octopod chromosomes and neural-related gene families. The single-nucleus transcriptomes of the supra-esophageal brain indicate their cellular heterogeneities and functional interactions with other tissues (i.e., gill), which provides a foundation for further octopod cerebral studies.

The molecular evolution of spermatogenesis across mammals

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1–6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Single-cell transcriptomics for the assessment of cardiac disease.

Cardiovascular disease is the leading cause of death globally. An advanced understanding of cardiovascular disease mechanisms is required to improve therapeutic strategies and patient risk stratification. State-of-the-art, large-scale, single-cell and single-nucleus transcriptomics facilitate the exploration of the cardiac cellular landscape at an unprecedented level, beyond its descriptive features, and can further our understanding of the mechanisms of disease and guide functional studies. In this Review, we provide an overview of the technical challenges in the experimental design of single-cell and single-nucleus transcriptomics studies, as well as a discussion of the type of inferences that can be made from the data derived from these studies. Furthermore, we describe novel findings derived from transcriptomics studies for each major cardiac cell type in both health and disease, and from development to adulthood. This Review also provides a guide to interpreting the exhaustive list of newly identified cardiac cell types and states, and highlights the consensus and discordances in annotation, indicating an urgent need for standardization. We describe advanced applications such as integration of single-cell data with spatial transcriptomics to map genes and cells on tissue and define cellular microenvironments that regulate homeostasis and disease progression. Finally, we discuss current and future translational and clinical implications of novel transcriptomics approaches, and provide an outlook of how these technologies will change the way we diagnose and treat heart disease.

Re-Analysis of Single-Nucleus Transcriptomics Reveals Diverse Dorsal Root Ganglia Macrophage Responses Following Peripheral Nerve Injury.

An increasing amount of evidence points to an important role of macrophages in peripheral nerve injury (PNI) and associated pain. Peripheral nerve macrophages facilitate the regeneration, while dorsal root ganglia (DRG) macrophages might propagate the injury after a PNI. These differences might be explained by various in vivo models of PNIs or non-uniform methodologies to phenotype the macrophages. Unbiased methods to phenotype macrophages using single whole cell or nucleus transcriptomics have been rarely applied on PNIs outside the nerves themselves. Here, we compare the effects of the transection or crush of the sciatic nerve and spinal nerve transection on the DRG macrophage phenotypes utilizing a publicly available single-nucleus transcriptomic DRG dataset. Our results demonstrate that unique and time-dependent DRG macrophage gene expression profiles were produced by the three PNI models with particular macrophage clusters being enriched that were dependent on the severity of the neuronal injury score. PNI associated DRG macrophages were not purely anti- or pro-inflammatory. These results suggest that various functions of DRG macrophage subtypes are carefully orchestrated upon a PNI. These findings open a new avenue for studying the DRG macrophage subtypes in PNIs and encourage further unbiased phenotyping efforts to better understand their relevance in neuropathic pain.

An atlas of late prenatal human neurodevelopment resolved by single-nucleus transcriptomics

Late prenatal development of the human neocortex encompasses a critical period of gliogenesis and cortical expansion. However, systematic single-cell analyses to resolve cellular diversity and gliogenic lineages of the third trimester are lacking. Here, we present a comprehensive single-nucleus RNA sequencing atlas of over 200,000 nuclei derived from the proliferative germinal matrix and laminating cortical plate of 15 prenatal, non-pathological postmortem samples from 17 to 41 gestational weeks, and 3 adult controls. This dataset captures prenatal gliogenesis with high temporal resolution and is provided as a resource for further interrogation. Our computational analysis resolves greater complexity of glial progenitors, including transient glial intermediate progenitor cell (gIPC) and nascent astrocyte populations in the third trimester of human gestation. We use lineage trajectory and RNA velocity inference to further characterize specific gIPC subpopulations preceding both oligodendrocyte (gIPC-O) and astrocyte (gIPC-A) lineage differentiation. We infer unique transcriptional drivers and biological pathways associated with each developmental state, validate gIPC-A and gIPC-O presence within the human germinal matrix and cortical plate in situ, and demonstrate gIPC states being recapitulated across adult and pediatric glioblastoma tumors.

Single-nucleus transcriptomics reveals adipose tissue-derived signalling contributing to cardiovascular disease in a model of type-2 diabetes

Single-nucleus transcriptomics reveals adipose tissue-derived signalling contributing to cardiovascular disease in a model of type-2 diabetes

Single-nucleus transcriptomics of IDH1- and TP53-mutant glioma stem cells displays diversified commitment on invasive cancer progenitors

Glioma is a devastating brain tumor with a high mortality rate attributed to the glioma stem cells (GSCs) possessing high plasticity. Marker mutations in isocitrate dehydrogenase type 1 (IDH1) and tumor protein 53 (TP53) are frequent in gliomas and impact the cell fate decisions. Understanding the GSC heterogeneity within IDH1- and TP53- mutant tumors may elucidate possible treatment targets. Here, we performed single-nucleus transcriptomics of mutant and wild-type glioma samples sorted for Sox2 stem cell marker. For the first time the rare subpopulations of Sox2 + IDH1- and TP53-mutant GSCs were characterized. In general, GSCs contained the heterogeneity root subpopulation resembling active neural stem cells capable of asymmetric division to quiescent and transit amplifying cell branches. Specifically, double-mutant GSCs revealed the commitment on highly invasive oligodendrocyte- and astroglia-like progenitors. Additionally, double-mutant GSCs displayed upregulated markers of collagen synthesis, altered lipogenesis and high migration, while wild-type GSCs expressed genes related to ATP production. Wild-type GSC root population was highly heterogeneous and lacked the signature marker expression, thus glioblastoma treatment should emphasize on establishing differentiation protocol directed against residual GSCs. For the more differentiated IDH1- and TP53-mutant gliomas we suggest therapeutic targeting of migration molecules, such as CD44.

Repurposable Drugs That Interact with Steroid Responsive Gene Targets for Inner Ear Disease.

Corticosteroids, oral or transtympanic, remain the mainstay for inner ear diseases characterized by hearing fluctuation or sudden changes in hearing, including sudden sensorineural hearing loss (SSNHL), Meniere's disease (MD), and autoimmune inner ear disease (AIED). Despite their use across these diseases, the rate of complete recovery remains low, and results across the literature demonstrates significant heterogeneity with respect to the effect of corticosteroids, suggesting a need to identify more efficacious treatment options. Previously, our group has cross-referenced steroid-responsive genes in the cochlea with published single-cell and single-nucleus transcriptome datasets to demonstrate that steroid-responsive differentially regulated genes are expressed in spiral ganglion neurons (SGN) and stria vascularis (SV) cell types. These differentially regulated genes represent potential druggable gene targets. We utilized multiple gene target databases (DrugBank, Pharos, and LINCS) to identify orally administered, FDA approved medications that potentially target these genes. We identified 42 candidate drugs that have been shown to interact with these genes, with an emphasis on safety profile, and tolerability. This study utilizes multiple databases to identify drugs that can target a number of druggable genes in otologic disorders that are commonly treated with steroids, providing a basis for establishing novel repurposing treatment trials.

Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Treatment-associated remodeling of the pancreatic cancer endothelium at single-cell resolution.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most treatment refractory and lethal malignancies. The diversity of endothelial cell (EC) lineages in the tumor microenvironment (TME) impacts the efficacy of antineoplastic therapies, which in turn remodel EC states and distributions. Here, we present a single-cell resolution framework of diverse EC lineages in the PDAC TME in the context of neoadjuvant chemotherapy, radiotherapy, and losartan. We analyzed a custom single-nucleus RNA-seq dataset derived from 37 primary PDAC specimens (18 untreated, 14 neoadjuvant FOLFIRINOX + chemoradiotherapy, 5 neoadjuvant FOLFIRINOX + chemoradiotherapy + losartan). A single-nucleus transcriptome analysis of 15,185 EC profiles revealed two state programs (ribosomal, cycling), four lineage programs (capillary, arterial, venous, lymphatic), and one program that did not overlap significantly with prior signatures but was enriched in pathways involved in vasculogenesis, stem-like state, response to wounding and hypoxia, and endothelial-to-mesenchymal transition (reactive EndMT). A bulk transcriptome analysis of two independent cohorts (n = 269 patients) revealed that the lymphatic and reactive EndMT lineage programs were significantly associated with poor clinical outcomes. While losartan and proton therapy were associated with reduced lymphatic ECs, these therapies also correlated with an increase in reactive EndMT. Thus, the development and inclusion of EndMT-inhibiting drugs (e.g., nintedanib) to a neoadjuvant chemoradiotherapy regimen featuring losartan and/or proton therapy may be most effective in depleting both lymphatic and reactive EndMT populations and potentially improving patient outcomes.

Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging.

Aging poses a major risk factor for cardiovascular diseases, the leading cause of death in the aged population. However, the cell type-specific changes underlying cardiac aging are far from being clear. Here, we performed single-nucleus RNA-sequencing analysis of left ventricles from young and aged cynomolgus monkeys to define cell composition changes and transcriptomic alterations across different cell types associated with age. We found that aged cardiomyocytes underwent a dramatic loss in cell numbers and profound fluctuations in transcriptional profiles. Via transcription regulatory network analysis, we identified FOXP1, a core transcription factor in organ development, as a key downregulated factor in aged cardiomyocytes, concomitant with the dysregulation of FOXP1 target genes associated with heart function and cardiac diseases. Consistently, the deficiency of FOXP1 led to hypertrophic and senescent phenotypes in human embryonic stem cell-derived cardiomyocytes. Altogether, our findings depict the cellular and molecular landscape of ventricular aging at the single-cell resolution, and identify drivers for primate cardiac aging and potential targets for intervention against cardiac aging and associated diseases.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Accelerated aging-related transcriptome alterations in neurovascular unit cells in the brain of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia with no effective therapies. Aging is a dominant risk factor for AD. The neurovascular unit (NVU) plays an important role in maintaining homeostasis of the brain microenvironment. The accelerated aging of NVU cells may directly impair NVU function and contribute to AD pathogenesis. However, the expression patterns of aging-related genes (AGs) in NVU cells of AD remain unclear. In this study, we performed single-nucleus transcriptome analysis of 61,768 nuclei from prefrontal cortical samples of patients with AD and normal control (NC) subjects. Eight main cell types were identified, including astrocytes, microglia, excitatory neurons, inhibitory neurons, oligodendrocytes, oligodendrocyte precursor cells, pericytes, and endothelial cells. Transcriptomic analysis identified the expression patterns of AGs in NVU cells of AD. Gene set enrichment analysis confirmed the key aging-associated cellular pathways enriched in microglia and oligodendrocytes. These aging-related transcriptomic changes in NVU were cross-validated using bulk transcriptome data. The least absolute shrinkage and selection operator regression method was used to select the crucial AGs most associated with AD: IGF1R, MXI1, RB1, PPARA, NFE2L2, STAT5B, FOS, PRKCD, YWHAZ, HTT, MAPK9, HSPA9, SDHC, PRKDC, and PDPK1. This 15-gene model performed well in discriminating AD from NC samples. Among them, IGF1R, MXI1, PPARA, YWHAZ, and MAPK9 strongly correlated with pathologic progression in AD, were identified as critical regulators of AD. Although most AGs showed similar trends of expression changes in different types of NVU cells in AD, certain AGs were expressed in a cell-specific manner. Our comprehensive analysis of brain NVU from patients with AD reveals previously unknown molecular changes associated with aging that may underlie the functional dysregulation of NVU, providing important insights for exploring potential cell-specific therapeutic targets to restore brain homeostasis in AD.

Reconstruction of the Global Polarity of an Early Spider Embryo by Single-Cell and Single-Nucleus Transcriptome Analysis.

Patterning along an axis of polarity is a fundamental step in the development of a multicellular animal embryo. In the cellular field of an early spider embryo, Hedgehog signaling operates to specify a “fuzzy” French-flag-like pattern along the primary axis, which is related to the future anterior–posterior (A–P) axis. However, details regarding the generation and development of a diversity of cell states based on the embryo polarity are not known. To address this issue, we applied single-cell RNA sequencing to the early spider embryo consisting of approximately 2,000 cells. Our results confirmed that this technique successfully detected 3 cell populations corresponding to the germ layers and some transient cell states. We showed that the data from dissociated cells had sufficient information for reconstruction of a correct global A–P polarity of the presumptive ectoderm, without clear segregation of specific cell states. This outcome is explained by the varied but differentially overlapping expression of Hedgehog-signal target genes and newly identified marker genes. We also showed that the data resources generated by the transcriptome analysis are applicable to a genome-wide search for genes whose expression is spatially regulated, based on the detection of pattern similarity. Furthermore, we performed single-nucleus RNA sequencing, which was more powerful in detecting emerging cell states. The single-cell and single-nucleus transcriptome techniques will help investigate the pattern-forming processes in the spider model system in an unbiased, comprehensive manner. We provided web-based resources of these transcriptome datasets for future studies of pattern formation and cell differentiation.

Intratumoral Heterogeneity and Immune Modulation in Lung Adenocarcinoma in Female Smokers and Never Smokers.

Single-cell analysis of healthy lung tissue and lung cancer reveals distinct tumor cell populations, including cells with differential immune modulating capacity between smokers and never smokers, which could guide future therapeutic strategies.

A single-cell transcriptomic atlas tracking the neural basis of division of labour in an ant superorganism

Ant colonies with permanent division of labour between castes and highly distinct roles of the sexes have been conceptualized to be superorganisms, but the cellular and molecular mechanisms that mediate caste/sex-specific behavioural specialization have remained obscure. Here we characterized the brain cell repertoire of queens, gynes (virgin queens), workers and males of Monomorium pharaonis by obtaining 206,367 single-nucleus transcriptomes. In contrast to Drosophila, the mushroom body Kenyon cells are abundant in ants and display a high diversity with most subtypes being enriched in worker brains, the evolutionarily derived caste. Male brains are as specialized as worker brains but with opposite trends in cell composition with higher abundances of all optic lobe neuronal subtypes, while the composition of gyne and queen brains remained generalized, reminiscent of solitary ancestors. Role differentiation from virgin gynes to inseminated queens induces abundance changes in roughly 35% of cell types, indicating active neurogenesis and/or programmed cell death during this transition. We also identified insemination-induced cell changes probably associated with the longevity and fecundity of the reproductive caste, including increases of ensheathing glia and a population of dopamine-regulated Dh31-expressing neurons. We conclude that permanent caste differentiation and extreme sex-differentiation induced major changes in the neural circuitry of ants.

Single-nucleus transcriptome analysis reveals disease- and regeneration-associated endothelial cells in white matter vascular dementia.

BackgroundVascular dementia (VaD) is the accumulation of vascular lesions in the subcortical white matter of the brain. These lesions progress and there is no direct medical therapy.AimsTo determine the specific cellular responses in VaD so as to provide molecular targets for therapeutic development.Materials and MethodsSingle‐nucleus transcriptome analysis was performed in human periventricular white matter (PVWM) samples of VaD and normal control (NC) subjects.ResultsDifferential analysis shows that cell type‐specific transcriptomic changes in VaD are associated with the disruption of specific biological processes, including angiogenesis, immune activation, axonal injury and myelination. Each cell type in the neurovascular unit within white matter has a specific alteration in gene expression in VaD. In a central cell type for this disease, subcluster analysis of endothelial cells (EC) indicates that VaD contains a disease‐associated EC subcluster that expresses genes associated with programmed cell death and a response to protein folding. Two other subpopulations of EC in VaD express molecular systems associated with regenerative processes in angiogenesis, and in axonal sprouting and oligodendrocyte progenitor cell maturation.ConclusionThis comprehensive molecular profiling of brain samples from patients with VaD reveals previously unknown molecular changes in cells of the neurovascular niche, and an attempt at regeneration in injured white matter.