Perivascular NOTCH3+ stem cells drive meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental. Resistance to radiotherapy is common in high-grade meningiomas and the cell types and signaling mechanisms that drive meningioma tumorigenesis and resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find that perivascular NOTCH3+ stem cells are conserved across meningiomas from humans, dogs, and mice. Integrating single-cell transcriptomics with lineage tracing and imaging approaches in genetically engineered mouse models and xenografts, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. To translate these findings to patients, we show that an antibody stabilizing the extracellular negative regulatory region of NOTCH3 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival.

Radiation-Induced Cellular Plasticity: A Strategy for Combatting Glioblastoma.

Glioblastoma is the deadliest brain cancer in adults and almost all patients succumb to the tumor. While surgery followed by chemo-radiotherapy significantly delays disease progression, these treatments do not lead to long-term tumor control and targeted therapies or biologics have so far failed to further improve survival. Utilizing a transient radiation-induced state of multipotency we used the adenylcyclase activator forskolin to alter the cellular fate of glioma cells in response to radiation. The combined treatment induced the expression of neuronal markers in glioma cells, reduced proliferation and led to a distinct gene expression profile. scRNAseq revealed that the combined treatment forced glioma cells into a microglia- and neuron-like phenotypes. In vivo this treatment led to a loss of glioma stem cells and prolonged median survival in mouse models of glioblastoma. Collectively, our data suggest that revisiting a differentiation therapy with forskolin in combination with radiation could lead to clinical benefit.

Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders.

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Profiling human brain vascular cells using single-cell transcriptomics and organoids.

Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.

Functional genomics and systems biology in human neuroscience.

Neuroscience research has entered a phase of key discoveries in the realm of neurogenomics owing to strong financial and intellectual support for resource building and tool development. The previous challenge of tissue heterogeneity has been met with the application of techniques that can profile individual cells at scale. Moreover, the ability to perturb genes, gene regulatory elements and neuronal activity in a cell-type-specific manner has been integrated with gene expression studies to uncover the functional underpinnings of the genome at a systems level. Although these insights have necessarily been grounded in model systems, we now have the opportunity to apply these approaches in humans and in human tissue, thanks to advances in human genetics, brain imaging and tissue collection. We acknowledge that there will probably always be limits to the extent to which we can apply the genomic tools developed in model systems to human neuroscience; however, as we describe in this Perspective, the neuroscience field is now primed with an optimal foundation for tackling this ambitious challenge. The application of systems-level network analyses to these datasets will facilitate a deeper appreciation of human neurogenomics that cannot otherwise be achieved from directly observable phenomena.

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus.

The development and diversity of neuronal subtypes in the human hypothalamus has been insufficiently characterized. To address this, we integrated transcriptomic data from 241,096 cells (126,840 newly generated) in the prenatal and adult human hypothalamus to reveal a temporal trajectory from proliferative stem cell populations to mature hypothalamic cell types. Iterative clustering of the adult neurons identified 108 robust transcriptionally distinct neuronal subtypes representing 10 hypothalamic nuclei. Pseudotime trajectories provided insights into the genes driving formation of these nuclei. Comparisons to single-cell transcriptomic data from the mouse hypothalamus suggested extensive conservation of neuronal subtypes despite certain differences in species-enriched gene expression. The uniqueness of hypothalamic neuronal lineages was examined developmentally by comparing excitatory lineages present in cortex and inhibitory lineages in ganglionic eminence, revealing both distinct and shared drivers of neuronal maturation across the human forebrain. These results provide a comprehensive transcriptomic view of human hypothalamus development through gestation and adulthood at cellular resolution.

LIF signaling regulates outer radial glial to interneuron fate during human cortical development.

Radial glial (RG) development is essential for cerebral cortex growth and organization. In humans, the outer radial glia (oRG) subtype is expanded and gives rise to diverse neurons and glia. However, the mechanisms regulating oRG differentiation are unclear. oRG cells express leukemia-inhibitory factor (LIF) receptors during neurogenesis, and consistent with a role in stem cell self-renewal, LIF perturbation impacts oRG proliferation in cortical tissue and organoids. Surprisingly, LIF treatment also increases the production of inhibitory interneurons (INs) in cortical cultures. Comparative transcriptomic analysis identifies that the enhanced IN population resembles INs produced in the caudal ganglionic eminence. To evaluate whether INs could arise from oRGs, we isolated primary oRG cells and cultured them with LIF. We observed the production of INs from oRG cells and an increase in IN abundance following LIF treatment. Our observations suggest that LIF signaling regulates the capacity of oRG cells to generate INs.

A Meta-Atlas of the Developing Human Cortex Identifies Modules Driving Cell Subtype Specification.

Human brain development requires the generation of hundreds of diverse cell types, a process targeted by recent single-cell transcriptomic profiling efforts. Through a meta-analysis of seven of these published datasets, we have generated 225 meta-modules - gene co-expression networks that can describe mechanisms underlying cortical development. Several meta-modules have potential roles in both establishing and refining cortical cell type identities, and we validated their spatiotemporal expression in primary human cortical tissues. These include meta-module 20, associated with FEZF2+ deep layer neurons. Half of meta-module 20 genes are putative FEZF2 targets, including TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical organoid experiments validated that both factors are necessary for deep layer neuron specification. Importantly, subtle manipulations of these factors drive slight changes in meta-module activity that cascade into strong differences in cell fate - demonstrating how of our meta-atlas can engender further mechanistic analyses of cortical fate specification.

Meningeal solitary fibrous tumor cell states phenocopy cerebral vascular development and homeostasis.

Meningeal solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that are associated with hematogenous metastasis, and the cell states and spatial transcriptomic architecture of SFTs are unknown. Here we use single-cell and spatial RNA sequencing to show SFTs are comprised of regionally distinct gene expression programs that resemble cerebral vascular development and homeostasis. Our results shed light on pathways underlying SFT biology in comparison to other central nervous system tumors and provide a framework for integrating single-cell and spatial transcriptomic data from human cancers and normal tissues.

NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors1-3. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental4,5. Resistance to radiotherapy is common in high-grade meningiomas6, and the cell types and signaling mechanisms driving meningioma tumorigenesis or resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find NOTCH3+ meningioma mural cells are conserved across meningiomas from humans, dogs, and mice. NOTCH3+ cells are restricted to the perivascular niche during meningeal development and homeostasis and in low-grade meningiomas but are expressed throughout high-grade meningiomas that are resistant to radiotherapy. Integrating single-cell transcriptomics with lineage tracing and imaging approaches across mouse genetic and xenograft models, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. An antibody stabilizing the extracellular negative regulatory region of NOTCH37,8 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival in preclinical models. In summary, our results identify a conserved cell type and signaling mechanism that underlie meningioma tumorigenesis and resistance to radiotherapy, revealing a new therapeutic vulnerability to treat meningiomas that are resistant to standard interventions.

Common molecular features of H3K27M DMGs and PFA ependymomas map to hindbrain developmental pathways.

Globally decreased histone 3, lysine 27 tri-methylation (H3K27me3) is a hallmark of H3K27-altered diffuse midline gliomas (DMGs) and group-A posterior fossa ependymomas (PFAs). H3K27-altered DMGs are largely characterized by lysine-to-methionine mutations in histone 3 at position 27 (H3K27M). Most PFAs overexpress EZH inhibitory protein (EZHIP), which possesses a region of similarity to the mutant H3K27M. Both H3K27M and EZHIP inhibit the function of the polycomb repressive complex 2 (PRC2) responsible for H3K27me3 deposition. These tumors often arise in neighboring regions of the brainstem and posterior fossa. In rare cases PFAs harbor H3K27M mutations, and DMGs overexpress EZHIP. These findings together raise the possibility that certain cell populations in the developing hindbrain/posterior fossa are especially sensitive to modulation of H3K27me3 states. We identified shared molecular features by comparing genomic, bulk transcriptomic, chromatin-based profiles, and single-cell RNA-sequencing (scRNA-seq) data from the two tumor classes. Our approach demonstrated that 1q gain, a key biomarker in PFAs, is prognostic in H3.1K27M, but not H3.3K27M gliomas. Conversely, Activin A Receptor Type 1 (ACVR1), which is associated with mutations in H3.1K27M gliomas, is overexpressed in a subset of PFAs with poor outcome. Despite diffuse H3K27me3 reduction, previous work shows that both tumors maintain genomic H3K27me3 deposition at select sites. We demonstrate heterogeneity in shared patterns of residual H3K27me3 for both tumors that largely segregated with inferred anatomic tumor origins and progenitor populations of tumor cells. In contrast, analysis of genes linked to H3K27 acetylation (H3K27ac)-marked enhancers showed higher expression in astrocytic-like tumor cells. Finally, common H3K27me3-marked genes mapped closely to expression patterns in the human developing hindbrain. Overall, our data demonstrate developmentally relevant molecular similarities between PFAs and H3K27M DMGs and support the overall hypothesis that deregulated mechanisms of hindbrain development are central to the biology of both tumors.

K27M in canonical and noncanonical H3 variants occurs in distinct oligodendroglial cell lineages in brain midline gliomas.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate.

Pathway-based approach reveals differential sensitivity to E2F1 inhibition in glioblastoma.

Analysis of tumor gene expression is an important approach for the classification and identification of therapeutic vulnerabilities. However, targeting glioblastoma (GBM) based on molecular subtyping has not yet translated into successful therapies. Here, we present an integrative approach based on molecular pathways to expose new potentially actionable targets. We used gene set enrichment analysis (GSEA) to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene signatures. We identified key targets that are predicted to be differentially activated between tumors and were functionally validated in a library of gliomasphere cultures. Resultant cluster-specific gene signatures associated not only with hallmarks of cell cycle and stemness gene expression, but also with cell-type specific markers and different cellular states of GBM. Several upstream regulators, such as PIK3R1 and EBF1 were differentially enriched in cells bearing stem cell like signatures and bear further investigation. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only a subset of gliomasphere cultures predicted to be E2F1 signaling dependent. Our in vivo work also validated the functional significance of E2F1 in tumor formation capacity in the predicted samples. E2F1 inhibition also differentially sensitized E2F1-dependent gliomasphere cultures to radiation treatment. Our findings indicate that this novel approach exploring cancer pathways highlights key therapeutic vulnerabilities for targeting GBM.

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Evaluation of advances in cortical development using model systems.

Compared with that of even the closest primates, the human cortex displays a high degree of specialization and expansion that largely emerges developmentally. Although decades of research in the mouse and other model systems has revealed core tenets of cortical development that are well preserved across mammalian species, small deviations in transcription factor expression, novel cell types in primates and/or humans, and unique cortical architecture distinguish the human cortex. Importantly, many of the genes and signaling pathways thought to drive human-specific cortical expansion also leave the brain vulnerable to disease, as the misregulation of these factors is highly correlated with neurodevelopmental and neuropsychiatric disorders. However, creating a comprehensive understanding of human-specific cognition and disease remains challenging. Here, we review key stages of cortical development and highlight known or possible differences between model systems and the developing human brain. By identifying the developmental trajectories that may facilitate uniquely human traits, we highlight open questions in need of approaches to examine these processes in a human context and reveal translatable insights into human developmental disorders.

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

Mounting evidence suggests human adult neurogenesis is unlikely.

In this issue of Neuron, Franjic et al. (2022) use a single-nuclei RNA sequencing approach that identified signatures of adult neurogenesis in mouse, pig, and macaque dentate gyrus, but not in humans, adding to a growing body of evidence that this process is likely lost in humans.

Cortical Cartography: Mapping Arealization Using Single-Cell Omics Technology.

The cerebral cortex derives its cognitive power from a modular network of specialized areas processing a multitude of information. The assembly and organization of these regions is vital for human behavior and perception, as evidenced by the prevalence of area-specific phenotypes that manifest in neurodevelopmental and psychiatric disorders. Generations of scientists have examined the architecture of the human cortex, but efforts to capture the gene networks which drive arealization have been hampered by the lack of tractable models of human neurodevelopment. Advancements in "omics" technologies, imaging, and computational power have enabled exciting breakthroughs into the molecular and structural characteristics of cortical areas, including transcriptomic, epigenomic, metabolomic, and proteomic profiles of mammalian models. Here we review the single-omics atlases that have shaped our current understanding of cortical areas, and their potential to fuel a new era of multi-omic single-cell endeavors to interrogate both the developing and adult human cortex.

An atlas of cortical arealization identifies dynamic molecular signatures.

The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood1. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that 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 approach, we find 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.