Dissecting the human leptomeninges at single-cell resolution.

Emerging evidence shows that the meninges conduct essential immune surveillance and immune defense at the brain border, and the dysfunction of meningeal immunity contributes to aging and neurodegeneration. However, no study exists on the molecular properties of cell types within human leptomeninges. Here, we provide single nuclei profiling of dissected postmortem leptomeninges from aged individuals. We detect diverse cell types, including unique meningeal endothelial, mural, and fibroblast subtypes. For immune cells, we show that most T cells express CD8 and bear characteristics of tissue-resident memory T cells. We also identify distinct subtypes of border-associated macrophages (BAMs) that display differential gene expressions from microglia and express risk genes for Alzheimer's Disease (AD), as nominated by genome-wide association studies (GWAS). We discover cell-type-specific differentially expressed genes in individuals with Alzheimer's dementia, particularly in fibroblasts and BAMs. Indeed, when cultured, leptomeningeal cells display the signature of ex vivo AD fibroblasts upon amyloid-ß treatment. We further explore ligand-receptor interactions within the leptomeningeal niche and computationally infer intercellular communications in AD. Thus, our study establishes a molecular map of human leptomeningeal cell types, providing significant insight into the border immune and fibrotic responses in AD.

Generation and molecular characterization of human pluripotent stem cell-derived pharyngeal foregut endoderm.

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs.

Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian Cells.

CRISPR/Cas9-based regulation of gene expression provides the scientific community with a new high-throughput tool to dissect the role of genes in molecular processes and cellular functions. Single-guide RNAs allow for recruitment of a nuclease-dead Cas9 protein and transcriptional Cas9-effector fusion proteins to specific genomic loci, thereby modulating gene expression. We describe the application of a CRISPR-Cas9 effector system from Streptococcus pyogenes for transcriptional regulation in mammalian cells resulting in activation or repression of transcription. We present methods for appropriate target site selection, sgRNA design, and delivery of dCas9 and dCas9-effector system components into cells through lentiviral transgenesis to modulate transcription.

Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.

Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules.

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders.