Cell Type-specific Profiles Research Articles

Cell Type-Specific Profiles and Developmental Trajectories of Transcriptomes in Primate Prefrontal Layer 3 Pyramidal Neurons: Implications for Schizophrenia.

In schizophrenia, impaired working memory is associated with transcriptome alterations in layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC). Distinct subtypes of L3PNs that send axonal projections to the DLPFC in the opposite hemisphere (callosal projection [CP] neurons) or the parietal cortex in the same hemisphere (ipsilateral projection [IP] neurons) play critical roles in working memory. However, how the transcriptomes of these L3PN subtypes might shift during late postnatal development when working memory impairments emerge in individuals later diagnosed with schizophrenia is not known. The aim of this study was to characterize and compare the transcriptome profiles of CP and IP L3PNs across developmental transitions from prepuberty to adulthood in macaque monkeys. The authors used retrograde labeling to identify CP and IP L3PNs in the DLPFC of prepubertal, postpubertal, and adult macaque monkeys, and used laser microdissection to capture these neurons for RNA sequencing. At all three ages, CP and IP L3PNs had distinct transcriptomes, with the number of genes differentially expressed between neuronal subtypes increasing with age. For IP L3PNs, age-related shifts in gene expression were most prominent between prepubertal and postpubertal animals, whereas for CP L3PNs such shifts were most prominent between postpubertal and adult animals. These findings demonstrate the presence of cell type-specific profiles and developmental trajectories of the transcriptomes of PPC-projecting IP and DLPFC-projecting CP L3PNs in monkey DLPFC. The evidence that IP L3PNs reach a mature transcriptome earlier than CP L3PNs suggests that these two subtypes differentially contribute to the maturation of working memory performance across late postnatal development and that they may be differentially vulnerable to the disease process of schizophrenia at specific stages of postnatal development.

Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues.

Mass spectrometry imaging (MSI) is a powerful technique for label-free spatial mapping of multiple classes of biomolecules in tissue sections. However, differences in desorption and ionization efficiency of different classes of molecules make it challenging to simultaneously map biomolecules at each omics layer in the same tissue sample. Herein, we present a correlative imaging method using nanospray desorption electrospray ionization (nano-DESI) MSI, which enables the spatial mapping of lipids, metabolites, peptides, and proteins with cellular-level spatial resolution in a single tissue section. We demonstrate the molecular profiling of specific cell types and identify truncated peptides in mouse pancreatic tissue. Distinct chemical gradients of peptides and lipids extending from endocrine cells to exocrine cells indicate their different roles in endocrine-exocrine crosstalk and intracellular signaling. The results underscore the power of the developed imaging approach for spatial multi-omics analysis that provides deep insights into cellular diversity and the intricate molecular interactions that occur within heterogenous biological tissues.

Cell type-specific regulation by different cytokinetic pathways in the early embryo.

Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.

Cell type-specific regulation by different cytokinetic pathways in the early embryo.

Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early 2-cell through 8-cell C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.

Inflammation in the tumor-adjacent lung as a predictor of clinical outcome in lung adenocarcinoma

Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.

Different activity patterns control various stages of Reelin synthesis in the developing neocortex.

Reelin is a large extracellular matrix protein abundantly expressed in the developing neocortex of mammals. During embryonic and early postnatal stages in mice, Reelin is secreted by a transient neuronal population, the Cajal-Retzius neurons (CRs), and is mostly known to insure the inside-out migration of neurons and the formation of cortical layers. During the first 2 postnatal weeks, CRs disappear from the neocortex and a subpopulation of GABAergic neurons takes over the expression of Reelin, albeit in lesser amounts. Although Reelin expression requires a tight regulation in a time- and cell-type specific manner, the mechanisms regulating the expression and secretion of this protein are poorly understood. In this study, we establish a cell-type specific profile of Reelin expression in the marginal zone of mice neocortex during the first 3 postnatal weeks. We then investigate whether electrical activity plays a role in the regulation of Reelin synthesis and/or secretion by cortical neurons during the early postnatal period. We show that increased electrical activity promotes the transcription of reelin via the brain-derived neurotrophic factor/TrkB pathway, but does not affect its translation or secretion. We further demonstrate that silencing the neuronal network promotes the translation of Reelin without affecting the transcription or secretion. We conclude that different patterns of activity control various stages of Reelin synthesis, whereas its secretion seems to be constitutive.

A single-cell atlas reveals shared and distinct immune responses and metabolic profiles in SARS-CoV-2 and HIV-1 infections.

Introduction: Within the inflammatory immune response to viral infection, the distribution and cell type-specific profiles of immune cell populations and the immune-mediated viral clearance pathways vary according to the specific virus. Uncovering the immunological similarities and differences between viral infections is critical to understanding disease progression and developing effective vaccines and therapies. Insight into COVID-19 disease progression has been bolstered by the integration of single-cell (sc)RNA-seq data from COVID-19 patients with data from related viruses to compare immune responses. Expanding this concept, we propose that a high-resolution, systematic comparison between immune cells from SARS-CoV-2 infection and an inflammatory infectious disease with a different pathophysiology will provide a more comprehensive picture of the viral clearance pathways that underscore immunological and clinical differences between infections. Methods: Using a novel consensus single-cell annotation method, we integrate previously published scRNA-seq data from 111,566 single PBMCs from 7 COVID-19, 10 HIV-1+, and 3 healthy patients into a unified cellular atlas. We compare in detail the phenotypic features and regulatory pathways in the major immune cell clusters. Results: While immune cells in both COVID-19 and HIV-1+ cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activity, and downregulated mitophagy. Discussion: Our results indicate that differential IFN-I signaling regulates the distinct immune responses in the two diseases, revealing insight into fundamental disease biology and potential therapeutic candidates.

Cell-type specific profiling of histone post-translational modifications in the adult mouse striatum

Epigenetic gene regulation in the heterogeneous brain remains challenging to decipher with current strategies. Bulk tissue analysis from pooled subjects reflects the average of cell-type specific changes across cell-types and individuals, which obscures causal relationships between epigenetic modifications, regulation of gene expression, and complex pathology. To address these limitations, we optimized a hybrid protocol, ICuRuS, for the isolation of nuclei tagged in specific cell-types and histone post translational modification profiling from the striatum of a single mouse. We combined affinity-based isolation of the medium spiny neuron subtypes, Adenosine 2a Receptor or Dopamine Receptor D1, with cleavage of histone-DNA complexes using an antibody-targeted micrococcal nuclease to release DNA complexes for paired end sequencing. Unlike fluorescence activated cell sorting paired with chromatin immunoprecipitation, ICuRuS allowed for robust epigenetic profiling at cell-type specific resolution. Our analysis provides a framework to understand combinatorial relationships between neuronal-subtype-specific epigenetic modifications and gene expression.

Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses

BackgroundIsolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. New methodTaking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. ResultsWe demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. Comparison with existing methodsOur method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. ConclusionsWe provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.

Repeated cocaine administration upregulates CB2 receptor expression in striatal medium-spiny neurons that express dopamine D1 receptors in mice.

Cannabinoid CB2 receptors (CB2R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB2R action remain unclear. We have previously reported that cocaine self-administration upregulates CB2R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB2R expression in striatal medium-spiny neuronsthat express dopamineD1 or D2 receptors (D1-MSNs, D2-MSNs) and microglia. Due to the concern of CB2R antibody specificity, we developed three mouse CB2-specific probes to detect CB2R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB2R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration ofheroin produced a dose-dependent reduction in striatal CB2 mRNA expression. RNAscope ISH assays detected CB2R mRNA in striatal D1- and D2-MSNs, not in microglia. We then used transgenic CX3CR1eGFP/+ microglia reporter mice and D1- or D2-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1eGFP/+,D1-MSNs, or D2-MSNs, respectively. We found that CB2R upregulation occurred mainly in D1-MSNs, not in D2-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB2R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB2R upregulation occurs mainly in D1-MSNs in the nucleus accumbens. Given the important role of D1-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB2Rs modulate cocaine action.

Hippocampal transcriptome profiling reveals common disease pathways in chronic hypoperfusion and aging.

Vascular dementia (VaD) is a progressive cognitive impairment of vascular etiology. VaD is characterized by cerebral hypoperfusion, increased blood-brain barrier permeability and white matter lesions. An increased burden of VaD is expected in rapidly aging populations. The hippocampus is particularly susceptible to hypoperfusion, and the resulting memory impairment may play a crucial role in VaD. Here we have investigated the hippocampal gene expression profile of young and old mice subjected to cerebral hypoperfusion by bilateral common carotid artery stenosis (BCAS). Our data in sham-operated young and aged mice reveal an age-associated decline in cerebral blood flow and differential gene expression. In fact, BCAS and aging caused broadly similar effects. However, BCAS-induced changes in hippocampal gene expression differed between young and aged mice. Specifically, transcriptomic analysis indicated that in comparison to young sham mice, many pathways altered by BCAS in young mice resembled those already present in sham aged mice. Over 30 days, BCAS in aged mice had minimal effect on either cerebral blood flow or hippocampal gene expression. Immunoblot analyses confirmed these findings. Finally, relative to young sham mice the cell type-specific profile of genes in both young BCAS and old sham animals further revealed common cell-specific genes. Our data provide a genetic-based molecular framework for hypoperfusion-induced hippocampal damage and reveal common cellular signaling pathways likely to be important in the pathophysiology of VaD.

Nuclei isolation of multiple brain cell types for omics interrogation.

We present a nuclei isolation protocol for genomic and epigenomic interrogation of multiple cell type populations in the human and rodent brain. The nuclei isolation protocol allows cell type-specific profiling of neurons, microglia, oligodendrocytes, and astrocytes, being compatible with fresh and frozen samples obtained from either resected or postmortem brain tissue. This 2-day procedure consists of tissue homogenization with fixation, nuclei extraction, and antibody staining followed by fluorescence-activated nuclei sorting (FANS) and does not require specialized skillsets. Cell type-specific nuclei populations can be used for downstream omic-scale sequencing applications with an emphasis on epigenomic interrogation such as histone modifications, transcription factor binding, chromatin accessibility, and chromosome architecture. The nuclei isolation protocol enables translational examination of archived healthy and diseased brain specimens through utilization of existing medical biorepositories.

Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue.

Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms.

Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation.

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from Drosophila midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system.

Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress.

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment.

Generation and Analysis of Pluripotent Stem Cell-Derived Cardiomyocytes and Endothelial Cells for High Content Screening Purposes.

Human-induced pluripotent stem cells (hiPSCs) and their differentiated derivatives became a new, promising source for in vitro screening techniques. Cell lines derived from healthy individuals can be applied for drug safety testing, while patient-derived cells provide a platform to model diseases in vitro and can be used as a tool for personalized medicine including specific drug efficacy testing and identification of new pharmacological targets as well as for tailoring pharmacological therapies. Efficient differentiation protocols yielding cardiomyocytes or endothelial cells derived from iPSCs have been developed recently. Phenotypic characterization and gene expression profiling of these derivatives can reveal clues for developmental and pathological questions. Moreover, functional analysis and cell-based assays using automated fluorescence imaging platform and high content analysis characterize cell type-specific profiles of hiPSC-derived cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC) at the cellular and subcellular levels. This can be utilized in a platform which can provide multiple endpoint profiles of candidate compounds.

Bioinformatics Approaches to Profile the Tumor Microenvironment for Immunotherapeutic Discovery.

In the microenvironment of a malignancy, tumor cells do not exist in isolation, but rather in a diverse ecosystem consisting not only of heterogeneous tumor-cell clones, but also normal cell types such as fibroblasts, vasculature, and an extensive pool of immune cells at numerous possible stages of activation and differentiation. This results in a complex interplay of diverse cellular signaling systems, where the immune cell component is now established to influence cancer progression and therapeutic response. It is experimentally difficult and laborious to comprehensively and systematically profile these distinct cell types from heterogeneous tumor samples in order to capitalize on potential therapeutic and biomarker discoveries. One emerging solution to address this challenge is to computationally extract cell-type specific information directly from bulk tumors. Such in silico approaches are advantageous because they can capture both the cell-type specific profiles and the tissue systems level of cell-cell interactions. Accurately and comprehensively predicting these patterns in tumors is an important challenge to overcome, not least given the success of immunotherapeutic drug treatment of several human cancers. This is especially challenging for subsets of closely related immune cell phenotypes with relatively small gene expression differences, which have critical functional distinctions. Here, we outline the existing and emerging novel bioinformatics strategies that can be used to profile the tumor immune landscape.

Chromatin state changes during neural development revealed by in vivo cell-type specific profiling

A key question in developmental biology is how cellular differentiation is controlled during development. While transitions between trithorax-group (TrxG) and polycomb-group (PcG) chromatin states are vital for the differentiation of ES cells to multipotent stem cells, little is known regarding the role of chromatin states during development of the brain. Here we show that large-scale chromatin remodelling occurs during Drosophila neural development. We demonstrate that the majority of genes activated during neuronal differentiation are silent in neural stem cells (NSCs) and occupy black chromatin and a TrxG-repressive state. In neurons, almost all key NSC genes are switched off via HP1-mediated repression. PcG-mediated repression does not play a significant role in regulating these genes, but instead regulates lineage-specific transcription factors that control spatial and temporal patterning in the brain. Combined, our data suggest that forms of chromatin other than canonical PcG/TrxG transitions take over key roles during neural development.

Cell Type-Specific Profiling of Chromatin Modifications and Associated Proteins.

Progression of a cell along a differentiation path is characterized by changes in gene expression profiles. Alterations of these transcriptional programs result from cell type-specific transcription factors that act in a dynamic chromatin environment. Understanding the precise contribution of these molecular factors during the differentiation process requires accessing specific cell types within a developing organ. This chapter describes a streamlined and alternative version of INTACT, a method enabling the isolation of specific cell populations by affinity-purification of tagged nuclei and the subsequent analysis of gene expression, transcription factor binding profiles, as well as chromatin state at a genome-wide scale. In particular, modifications of the nuclei isolation, capture, and purification procedures are proposed that improve time scale, yield, and purity. In addition, the combination of different tags enables the analysis of distinct cell populations from a single transgenic line and the subtractive purification of subpopulations of cells, including those for which no specific promoter is available. Finally, we describe a chromatin immunoprecipitation protocol that has been successfully used to profile histone modifications and other chromatin-associated proteins such as RNA Polymerase II in different cell populations of the Arabidopsis root, including the quiescent center of the stem cell niche.