Biology subjects

Kriegstein, A. R.

Publications and source records attributed to Kriegstein, A. R..

13 recordsLinked to original sources

bioRxiv2026

Scalable high-fidelity human vascularized cortical assembloids recapitulate neurovascular co-development and cell specialization

Human cortical development involves the coupling of neurogenesis and cerebrovascular growth. However, interactions between neural and vascular cells are largely missing in most brain organoids, which are crucial models for studying neurodevelopment and disease. Here, we establish vascularized cortical assembloids (vCAs) by fusing mesoderm-derived vascular organoids (VOs) with cortical organoids (COs). vCAs self-assemble lumenized networks of endothelial cells, pericytes, and perivascular fibroblasts that acquire blood-brain barrier (BBB) specialization and arteriovenous specification in vitro. Single-cell RNA-sequencing and immunofluorescence imaging revealed that vascularization improves neuroepithelial architecture, reduces hypoxia and apoptosis, expands cortical progenitor pools, and enhances neuronal maturation and connectivity compared with COs. Moreover, atlas-level integration with human neurodevelopmental tissue and cross-protocol benchmarking demonstrate superior transcriptional concordance, especially for vascular cell and glial populations, relative to existing approaches. This scalable and reproducible platform improves fidelity and throughput for modeling human neurovascular co-development and enables systematic studies across brain regions and diseases using engineered or patient-derived iPSCs.

developmental biology↗

bioRxiv2024

Molecular and spatial analysis of ganglion cells on retinal flatmounts: diversity, topography, and perivascularity

Diverse retinal ganglion cells (RGCs) transmit distinct visual features from the eye to the brain. Recent studies have categorized RGCs into 45 types in mice based on transcriptomic profiles, showing strong alignment with morphological and electrophysiological properties. However, little is known about how these types are spatially arranged on the two-dimensional retinal surface--an organization that influences visual encoding--and how their local microenvironments impact development and neurodegenerative responses. To address this gap, we optimized a workflow combining imaging-based spatial transcriptomics (MERFISH) and immunohistochemical co-staining on thin flatmount retinal sections. We used computational methods to register en face somata distributions of all molecularly defined RGC types. More than 75% (34/45) of types exhibited non-uniform distributions, likely reflecting adaptations of the retinas anatomy to the animals visual environment. By analyzing the local neighborhoods of each cell, we identified perivascular RGCs located near blood vessels. Seven RGC types are enriched in the perivascular niche, including members of intrinsically photosensitive RGC (ipRGC) and direction-selective RGC (DSGC) subclasses. Orthologous human RGC counterparts of perivascular types - Melanopsin-enriched ipRGCs and ON DSGCs - were also proximal to blood vessels, suggesting their perivascularity may be evolutionarily conserved. Following optic nerve crush in mice, the perivascular M1-ipRGCs and ON DSGCs showed preferential survival, suggesting that proximity to blood vessels may render cell-extrinsic neuroprotection to RGCs through an mTOR-independent mechanism. Overall, our work offers a resource characterizing the spatial profiles of RGC types, enabling future studies of retinal development, physiology, and neurodegeneration at individual neuron type resolution across the two-dimensional space.

neuroscience↗

bioRxiv2024

Fate plasticity of interneuron specification

The generation of neuronal subtypes in the mammalian central nervous system is driven by competing genetic programs. The medial ganglionic eminence (MGE) gives rise to two major cortical interneuron (cIN) populations, marked by Somatostatin (Sst) and Parvalbumin (Pvalb), which develop on different timelines. The extent to which external signals influence these identities remains poorly understood. Pvalb-positive cINs are particularly important for regulating cortical circuits through strong perisomatic inhibition, yet they have been difficult to model in vitro. Here we investigated the role of the environment in shaping and maintaining Pvalb cINs. We grafted mouse MGE progenitors into a variety of 2D and 3D co-culture models, including mouse and human cortical, MGE, and thalamic systems with dissociated cells, organoids, organotypic cultures, and conditioned media. Across models, we observed distinct proportions of Sst- and Pvalb-positive cIN descendants. Strikingly, grafting MGE progenitors into 3D human, but not mouse, corticogenesis models led to efficient, non-autonomous differentiation of Pvalb-positive cINs. This differentiation was characterized by upregulation of Pvalb maturation markers, downregulation of Sst-specific markers, and the formation of perineuronal nets. Furthermore, lineage-traced postmitotic Sst-positive cINs, when grafted onto human cortical models, also upregulated Pvalb expression. These results reveal an unexpected level of fate plasticity in MGE-derived cINs, demonstrating that their identities can be dynamically shaped by the surrounding environment.

neuroscience↗

bioRxiv2024

Spatial 3D genome organization controls the activity of bivalent chromatin during human neurogenesis

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1-5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery - not the presence of H3K27me3 - is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.

neuroscience↗

bioRxiv2024

Molecular and cellular dynamics of the developing human neocortex at single-cell resolution

The development of the human neocortex is a highly dynamic process and involves complex cellular trajectories controlled by cell-type-specific gene regulation1. Here, we collected paired single-nucleus chromatin accessibility and transcriptome data from 38 human neocortical samples encompassing both the prefrontal cortex and primary visual cortex. These samples span five main developmental stages, ranging from the first trimester to adolescence. In parallel, we performed spatial transcriptomic analysis on a subset of the samples to illustrate spatial organization and intercellular communication. This atlas enables us to catalog cell type-, age-, and area-specific gene regulatory networks underlying neural differentiation. Moreover, combining single-cell profiling, progenitor purification, and lineage-tracing experiments, we have untangled the complex lineage relationships among progenitor subtypes during the transition from neurogenesis to gliogenesis in the human neocortex. We identified a tripotential intermediate progenitor subtype, termed Tri-IPC, responsible for the local production of GABAergic neurons, oligodendrocyte precursor cells, and astrocytes. Remarkably, most glioblastoma cells resemble Tri-IPCs at the transcriptomic level, suggesting that cancer cells hijack developmental processes to enhance growth and heterogeneity. Furthermore, by integrating our atlas data with large-scale GWAS data, we created a disease-risk map highlighting enriched ASD risk in second-trimester intratelencephalic projection neurons. Our study sheds light on the gene regulatory landscape and cellular dynamics of the developing human neocortex.

neuroscience↗

bioRxiv2022

A cross-species proteomic map of synapse development reveals neoteny during human postsynaptic density maturation

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood. Here, we generated a cross-species proteomic map of synapse development in the human, macaque, and mouse neocortex. By tracking the changes of >1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we found that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that the human PSD matures about two to three times slower than other species and contains higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of the RhoGEF signaling in human neurons delays the morphological maturation of dendritic spines and the functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Together, our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

neuroscience↗

bioRxiv2021

An Atlas of Cortical Arealization Identifies Dynamic Molecular Signatures

The human brain is subdivided into distinct anatomical structures. The neocortex, one of these structures, enables higher-order sensory, associative, and cognitive functions, and in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish an early blueprint for the specification of brain regions and cortical areas. Furthermore, recent studies have uncovered distinct transcriptomic signatures between opposing poles of the developing neocortex1. However, how early, broad developmental patterns result in finer and more discrete spatial differences across the adult human brain remains poorly understood2. Here, we use single-cell RNA-sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Our data reveal that distinct cell subtypes are predominantly brain-structure specific. Within the neocortex, we find that even early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization (smFISH) approach, we validated the expression pattern of area-specific neuronal genes and also discover that laminar gene expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

developmental biology↗

bioRxiv2021

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

neuroscience↗

bioRxiv2020

mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex

Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease.

neuroscience↗

bioRxiv2020

Heterogeneity of Neuroepithelial Cells and Radial Glia

The human cortex is comprised of diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain, and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

developmental biology↗

bioRxiv2018

Regulation of Cell-Type-Specific Transcriptomes by miRNA Networks During Human Brain Development

MicroRNAs (miRNAs) regulate many cellular events by regulating hundreds of mRNA transcripts. However, it is unclear how miRNA-mRNA interactions are contextualized into the framework of transcriptional heterogeneity among closely related cells of the developing human brain. By combining the multiple complementary approaches, AGO2-HITS-CLIP, single-cell profiling and bipartite network analysis, we show that the miRNA-mRNA network operates as functional modules related to cell-type identities and undergo dynamic transitions during brain development.

developmental biology↗

bioRxiv2017

Saturating Single-Cell Datasets

High throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. Efficient generation of such an atlas will depend on sufficient sampling of the diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals. To examine the relationship between cell number and transcriptional heterogeneity in the context of unbiased cell type classification, we explicitly explored the population structure of a publically available 1.3 million cell dataset from the E18.5 mouse brain. We propose a computational framework for inferring the saturation point of cluster discovery in a single cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a \"complexity index\", which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells (20,000). Together, these findings suggest that most of the biologically interpretable insights from the 1.3 million cells can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high \"cellular coverage\", the much anticipated cell atlasing studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage.\n\nRecent efforts seek to create a comprehensive cell atlas of the human body1,2 Current technology, however, makes it precipitously expensive to perform analysis of every cell. Therefore, designing effective sampling strategies be critical to generate a working atlas in an efficient, cost-effective, and streamlined manner. The advent of single cell and single nucleus mRNA sequencing (RNAseq) in droplet format3,4 now enables large scale sampling of cells from any tissue, and a recently released publicly available dataset of 1.3 million single cells from the E18.5 mouse brain generated with the 10X Chromium5 provides an opportunity to explore the relationship between population structure and the number of sampled cells necessary to reveal the underlying diversity of cell types. Here, we present a framework for how researchers can evaluate whether a dataset has reached saturation, and we estimate how many cells would be required to generate an atlas of the sample analyzed here. This framework can be applied to any organ or cell type specific atlas for any organism.

bioinformatics↗

bioRxiv2017

Limited contribution of rare, noncoding variation to autism spectrum disorder from sequencing of 2,076 genomes in quartet families

Genomic studies to date in autism spectrum disorder (ASD) have largely focused on newly arising mutations that disrupt protein coding sequence and strongly influence risk. We evaluate the contribution of noncoding regulatory variation across the size and frequency spectrum through whole genome sequencing of 519 ASD cases, their unaffected sibling controls, and parents. Cases carry a small excess of de novo (1.02-fold) noncoding variants, which is not significant after correcting for paternal age. Assessing 51,801 regulatory classes, no category is significantly associated with ASD after correction for multiple testing. The strongest signals are observed in coding regions, including structural variation not detected by previous technologies and missense variation. While rare noncoding variation likely contributes to risk in neurodevelopmental disorders, no category of variation has impact equivalent to loss-of-function mutations. Average effect sizes are likely to be smaller than that for coding variation, requiring substantially larger samples to quantify this risk.

genomics↗