HIV-1 activates oxidative phosphorylation in infected CD4 T cells in a human tonsil explant model.

Introduction: Human immunodeficiency virus type 1 (HIV-1) causes a chronic, incurable infection leading to immune activation and chronic inflammation in people with HIV-1 (PWH), even with virologic suppression on antiretroviral therapy (ART). The role of lymphoid structures as reservoirs for viral latency and immune activation has been implicated in chronic inflammation mechanisms. Still, the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue remain unexplored. Methods: In this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1 ex vivo. We performed single-cell RNA sequencing (scRNA-seq) to analyze the cell types represented in the tissue and to investigate the impact of infection on gene expression profiles and inflammatory signaling pathways. Results: Our analysis revealed that infected CD4+ T cells exhibited upregulation of genes associated with oxidative phosphorylation. Furthermore, macrophages exposed to the virus but uninfected showed increased expression of genes associated with the NLRP3 inflammasome pathway. Discussion: These findings provide valuable insights into the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue. The activation of oxidative phosphorylation in infected CD4+ T cells and the proinflammatory response in macrophages may contribute to the chronic inflammation observed in PWH despite ART. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to eradicate HIV-1 infection in PWH.

A distal lung organoid model to study interstitial lung disease, viral infection and human lung development.

Organoids have been an exciting advancement in stem cell research. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids. This protocol recapitulates lung development by sequentially specifying human pluripotent stem cells to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids and finally lung organoids. The organoids take ~40 d to generate and can be maintained more than 180 d, while progressively maturing up to a stage consistent with the second trimester of human gestation. They are unique because of their branching morphology, the near absence of non-lung endodermal lineages, presence of mesenchyme and capacity to recapitulate interstitial lung diseases. This protocol can be performed by anyone familiar with cell culture techniques, is conducted in serum-free conditions and does not require lineage-specific reporters or enrichment steps. We also provide a protocol for the generation of single-cell suspensions for single-cell RNA sequencing.

Non-additive effects of schizophrenia risk genes reflect convergent downstream function.

Genetic studies of schizophrenia (SCZ) reveal a complex polygenic risk architecture comprised of hundreds of risk variants, the majority of which are common in the population at-large and confer only modest increases in disorder risk. Precisely how genetic variants with individually small predicted effects on gene expression combine to yield substantial clinical impacts in aggregate is unclear. Towards this, we previously reported that the combinatorial perturbation of four SCZ risk genes ("eGenes", whose expression is regulated by common variants) resulted in gene expression changes that were not predicted by individual perturbations, being most non-additive among genes associated with synaptic function and SCZ risk. Now, across fifteen SCZ eGenes, we demonstrate that non-additive effects are greatest within groups of functionally similar eGenes. Individual eGene perturbations reveal common downstream transcriptomic effects ("convergence"), while combinatorial eGene perturbations result in changes that are smaller than predicted by summing individual eGene effects ("sub-additive effects"). Unexpectedly, these convergent and sub-additive downstream transcriptomic effects overlap and constitute a large proportion of the genome-wide polygenic risk score, suggesting that functional redundancy of eGenes may be a major mechanism underlying non-additivity. Single eGene perturbations likewise fail to predict the magnitude or directionality of cellular phenotypes resulting from combinatorial perturbations. Overall, our results indicate that polygenic risk cannot be extrapolated from experiments testing one risk gene at a time and must instead be empirically measured. By unravelling the interactions between complex risk variants, it may be possible to improve the clinical utility of polygenic risk scores through more powerful prediction of symptom onset, clinical trajectory, and treatment response, or to identify novel targets for therapeutic intervention.

Human pluripotent stem cell-derived respiratory airway progenitors generate alveolar epithelial cells and recapitulate features of idiopathic pulmonary fibrosis.

Human lungs contain unique cell populations in distal respiratory airways (RAs). These populations accumulate in patients with lung injury, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Their lineage potentials and roles are unknown, however. As they are absent in rodents, deeper understanding of these cells requires a human in vitro model. Here we report the generation from human pluripotent stem cells (hPSCs) of expandable spheres (induced respiratory airway progenitors (iRAPs)) consisting of all RA-associated cell types. iRAPs could differentiate into type 1 (AT1) and type 2 alveolar (AT2) epithelial cells in defined conditions, showing that alveolar cells can be derived from RAs. iRAPs with deletion of HPS1, which causes pulmonary fibrosis in humans, display defects that are hallmarks of IPF, indicating involvement of intrinsic dysfunction of RA-associated cells in IPF. iRAPs thus provide a model to gain insight into human lung regeneration and into pathogenesis of IPF.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Chromatin profiling in human neurons reveals aberrant roles for histone acetylation and BET family proteins in schizophrenia.

Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications (PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide 'reader' of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons.

We describe a CRISPR inhibition (CRISPRi) protocol to repress endogenous gene expression (e.g., ATP6V1A) in human induced pluripotent stem cell-derived NGN2-induced glutamatergic neurons. CRISPRi enables efficient and precise gene repression of one or multiple target genes via delivering gRNA(s) to direct a dCas9-KRAB fusion protein to the gene(s) of interest. This protocol can also be adapted for gene activation and high-throughput gene manipulation, allowing assessment of the transcriptomic and phenotypic impact of candidate gene(s) associated with neurodevelopment or brain disease. For complete details on the use and execution of this protocol, please refer to Ho et al. (2017) and Wang et al. (2021).

Analysis framework and experimental design for evaluating synergy-driving gene expression.

The mechanisms by which genetic risk variants interact with each other, as well as environmental factors, to contribute to complex genetic disorders remain unclear. We describe in detail our recently published approach to resolve distinct additive and synergistic transcriptomic effects after combinatorial manipulation of genetic variants and/or chemical perturbagens. Although first developed for CRISPR-based perturbation studies of isogenic human induced pluripotent stem cell-derived neurons, our methodology can be broadly applied to any RNA sequencing dataset, provided that raw read counts are available. Whereas other differential expression analyses reveal the effect of individual perturbations, here we specifically query interactions between two or more perturbagens, resolving the extent of non-additive (synergistic) interactions between perturbations. We discuss the careful experimental design required to resolve synergistic effects and considerations of statistical power and how to quantify observed synergy between experiments. Additionally, we speculate on potential future applications and explore the obvious limitations of this approach. Overall, by interrogating the effect of independent factors, alone and in combination, our analytic framework and experimental design facilitate the discovery of convergence and synergy downstream of gene and/or treatment perturbations hypothesized to contribute to complex diseases. We think that this protocol can be successfully applied by any scientist with bioinformatic skills and basic proficiency in the R programming language. Our computational pipeline ( https://github.com/nadschro/synergy-analysis ) is straightforward, does not require supercomputing support and can be conducted in a single day upon completion of RNA sequencing experiments.

Transformative Network Modeling of Multi-omics Data Reveals Detailed Circuits, Key Regulators, and Potential Therapeutics for Alzheimer's Disease.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Integration of CRISPR-engineering and hiPSC-based models of psychiatric genomics.

Neuropsychiatric disorders are highly heritable polygenic disorders arising from the complex interplay of highly penetrant rare variants and common variants of small effect. There is a large index of comorbidity and shared genetic risk between disorders, reflecting the pleiotropy of individual variants as well as predicted downstream pathway-level convergence. Importantly, the mechanism(s) through which psychiatric disease-associated variants interact to contribute to disease risk remains unknown. Human induced pluripotent stem cell (hiPSC)-based models are increasingly useful for the systematic study of the complex genetics associated with brain diseases, particularly when combined with CRISPR-mediated genomic engineering, which together facilitate isogenic comparisons of defined neuronal cell types. In this review, we discuss the latest CRISPR technologies and consider how they can be successfully applied to the functional characterization of the growing list genetic variants linked to psychiatric disease.

Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models.

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

Neuronal impact of patient-specific aberrant NRXN1α splicing.

NRXN1 undergoes extensive alternative splicing, and non-recurrent heterozygous deletions in NRXN1 are strongly associated with neuropsychiatric disorders. We establish that human induced pluripotent stem cell (hiPSC)-derived neurons well represent the diversity of NRXN1α alternative splicing observed in the human brain, cataloguing 123 high-confidence in-frame human NRXN1α isoforms. Patient-derived NRXN1+/- hiPSC-neurons show a greater than twofold reduction in half of the wild-type NRXN1α isoforms and express dozens of novel isoforms from the mutant allele. Reduced neuronal activity in patient-derived NRXN1+/- hiPSC-neurons is ameliorated by overexpression of individual control isoforms in a genotype-dependent manner, whereas individual mutant isoforms decrease neuronal activity levels in control hiPSC-neurons. In a genotype-dependent manner, the phenotypic impact of patient-specific NRXN1+/- mutations can occur through a reduction in wild-type NRXN1α isoform levels as well as the presence of mutant NRXN1α isoforms.

Synergistic effects of common schizophrenia risk variants.

The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.

New considerations for hiPSC-based models of neuropsychiatric disorders.

The development of human-induced pluripotent stem cells (hiPSCs) has made possible patient-specific modeling across the spectrum of human disease. Here, we discuss recent advances in psychiatric genomics and post-mortem studies that provide critical insights concerning cell-type composition and sample size that should be considered when designing hiPSC-based studies of complex genetic disease. We review recent hiPSC-based models of SZ, in light of our new understanding of critical power limitations in the design of hiPSC-based studies of complex genetic disorders. Three possible solutions are a movement towards genetically stratified cohorts of rare variant patients, application of CRISPR technologies to engineer isogenic neural cells to study the impact of common variants, and integration of advanced genetics and hiPSC-based datasets in future studies. Overall, we emphasize that to advance the reproducibility and relevance of hiPSC-based studies, stem cell biologists must contemplate statistical and biological considerations that are already well accepted in the field of genetics. We conclude with a discussion of the hypothesis of biological convergence of disease-through molecular, cellular, circuit, and patient level phenotypes-and how this might emerge through hiPSC-based studies.

Neuron-specific signatures in the chromosomal connectome associated with schizophrenia risk.

To explore the developmental reorganization of the three-dimensional genome of the brain in the context of neuropsychiatric disease, we monitored chromosomal conformations in differentiating neural progenitor cells. Neuronal and glial differentiation was associated with widespread developmental remodeling of the chromosomal contact map and included interactions anchored in common variant sequences that confer heritable risk for schizophrenia. We describe cell type-specific chromosomal connectomes composed of schizophrenia risk variants and their distal targets, which altogether show enrichment for genes that regulate neuronal connectivity and chromatin remodeling, and evidence for coordinated transcriptional regulation and proteomic interaction of the participating genes. Developmentally regulated chromosomal conformation changes at schizophrenia-relevant sequences disproportionally occurred in neurons, highlighting the existence of cell type-specific disease risk vulnerabilities in spatial genome organization.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice.

BACKGROUND: The GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs. RESULTS: We have generated a Gata6(H2B-Venus) knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution. CONCLUSIONS: Expression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6(H2B-Venus/H2B-Venus) homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.

Lineage specificity of primary cilia in the mouse embryo.

Primary cilia are required for vertebrate cells to respond to specific intercellular signals. Here we define when and where primary cilia appear in the mouse embryo using a transgenic line that expresses ARL13B-mCherry in cilia and Centrin 2-GFP in centrosomes. Primary cilia first appear on cells of the epiblast at E6.0 and are subsequently present on all derivatives of the epiblast. In contrast, extraembryonic cells of the visceral endoderm and trophectoderm lineages have centrosomes but no cilia. Stem cell lines derived from embryonic lineages recapitulate the in vivo pattern: epiblast stem cells are ciliated, whereas trophoblast stem cells and extraembryonic endoderm (XEN) stem cells lack cilia. Basal bodies in XEN cells are mature and can form cilia when the AURKA-HDAC6 cilium disassembly pathway is inhibited. The lineage-dependent distribution of cilia is stable throughout much of gestation, defining which cells in the placenta and yolk sac are able to respond to Hedgehog ligands.

GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst.

Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.