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.
In this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1
. 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.
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.
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.
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).
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•CRISPRi enables efficient and precise gene repression of one or multiple target genes•This protocol describes gene repression in hiPSC-derived NGN2 neurons•This protocol can be adapted for gene activation and high-throughput gene manipulation
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.
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.
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.
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.
Schizophrenia (SZ) is a highly heritable neuropsychiatric disorder. Nevertheless, the functional mechanisms through which the large contribution of common genetic variation underlies this disorder ...remain unclear. GWAS and eQTL analyses have previously identified candidate genes, whose expression is affected by common SZ risk variants (SZ‐eQTL genes). We established a genetics‐driven hiPSC‐based approach for the prioritization and functional validation of common variants and genes associated with SZ.
Using this strategy, we prioritized one putative causal SZ‐eQTL SNP and five SZ‐eQTL genes for functional evaluation. By integrating CRISPR‐mediated gene editing, activation and repression technologies, our isogenic hiPSC‐based neuronal platform recapitulated genotype‐dependent gene expression differences from post‐mortem brains in SNP‐edited hiPSC‐neurons, identified evidence of molecular convergence downstream of common variant perturbations and uncovered specific pre‐ and post‐synaptic neuronal deficits associated with the SZ‐eQTL gene SNAP91.
We demonstrate a systematic and comprehensive strategy to interpret and evaluate the growing number of SZ‐associated variants and genes across neural cell types and genetic backgrounds, which will be applicable to other complex genetic disorders driven in large part by common variation.
Ultimately, our objective is to understand the cell‐type specific contributions of SZ risk variants to disease predisposition.
Support or Funding Information
This work was partially supported by National Institute of Health (NIH) grants R01 MH101454 (K.J.B.), R01 MH106056 (K.J.B.) and R01 MH109897 (P.S. and K.J.B.)
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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.
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.
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.
15 SCZ eGenes were selected for perturbation on the basis of their purported functional role: 5 eGenes involved in synaptic function, 5 transcriptional regulators and a mixed set comprised of 5 eGenes with multiple functional roles. Using using a combination of CRISPR activation and RNA interference, we performed perturbations of these 15 eGenes in hiPSC-derived cultures of excitatory neurons, individually and jointly within each functional set. The impact and interaction of these eGene perturbations on cellular and synaptic phenotypes was assessed using a combination of bulk RNA-seq, multi-electrode array recording and high-throughput imaging.
Here, 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.
At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells ...derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2-3 weeks.
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