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.

Individual human cortical progenitors can produce excitatory and inhibitory neurons.

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes-excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereas cortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called 'single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships' (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of cortically born GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex.

Tropism of SARS-CoV-2 for 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. Neurological symptoms, which range in severity, accompany as many as one-third 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 stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

A Case of Neonatal Lupus Presenting with Myocardial Dysfunction in the Absence of Congenital Heart Block (CHB): Clinical Management and Brief Literature Review of Neonatal Cardiac Lupus.

Neonatal lupus (NLE) is a rare acquired autoimmune disorder caused by transplacental passage of maternal autoantibodies to Sjogren's Syndrome A or B (SSA-SSB) autoantigens (Vanoni et al. in Clin Rev Allerg Immunol 53:469-476, 2017) which target fetal and neonatal tissues for immune destruction. The cardiac trademark of NLE is autoimmune heart block, which accounts for more than 80% of cases of complete atrioventricular heart block (AVB) in newborns with a structurally normal heart (Martin in Cardiol Young 24: 41-46, 2014). NLE presenting with cardiac alterations not involving rhythm disturbances are described in the literature, but they are rare. Here, we report a case of a neonate with high anti-SSA antibodies who developed severe ventricular dysfunction in the absence of rhythm abnormalities, endocardial fibroelastosis, and dilated cardiomyopathy (Trucco et al. in J Am Coll Cardiol 57:715-723, https://doi.org/10.1016/j.jacc.2010.09.044 , 2011), the most common cardiac presentations of NLE. The patient developed severe multiorgan dysfunction syndrome that required prolonged critical care support but fully recovered and was discharged home. We highlight the unusual clinical features of this NLE case and the importance of timely treatment of NLE allowing complete recovery of a critically ill neonate.

Regional and stage-specific effects of prospectively purified vascular cells on the adult V-SVZ neural stem cell lineage.

Adult neural stem cells reside in specialized niches. In the ventricular-subventricular zone (V-SVZ), quiescent neural stem cells (qNSCs) become activated (aNSCs), and generate transit amplifying cells (TACs), which give rise to neuroblasts that migrate to the olfactory bulb. The vasculature is an important component of the adult neural stem cell niche, but whether vascular cells in neurogenic areas are intrinsically different from those elsewhere in the brain is unknown. Moreover, the contribution of pericytes to the neural stem cell niche has not been defined. Here, we describe a rapid FACS purification strategy to simultaneously isolate primary endothelial cells and pericytes from brain microregions of nontransgenic mice using CD31 and CD13 as surface markers. We compared the effect of purified vascular cells from a neurogenic (V-SVZ) and non-neurogenic brain region (cortex) on the V-SVZ stem cell lineage in vitro. Endothelial and pericyte diffusible signals from both regions differentially promote the proliferation and neuronal differentiation of qNSCs, aNSCs, and TACs. Unexpectedly, diffusible cortical signals had the most potent effects on V-SVZ proliferation and neurogenesis, highlighting the intrinsic capacity of non-neurogenic vasculature to support stem cell behavior. Finally, we identify PlGF-2 as an endothelial-derived mitogen that promotes V-SVZ cell proliferation. This purification strategy provides a platform to define the functional and molecular contribution of vascular cells to stem cell niches and other brain regions under different physiological and pathological states.

A Vascular-Centric Approach to Autism Spectrum Disorders.

Brain development and function are highly reliant on adequate establishment and maintenance of vascular networks. Early impairments in vascular health can impact brain maturation and energy metabolism, which may lead to neurodevelopmental anomalies. Our recent work not only provides novel insights into the development of cerebrovascular networks but also emphasizes the importance of their well-being for proper brain maturation. In particular, we have demonstrated that endothelial dysfunction in autism spectrum disorders (ASD) mouse models is causally related to altered behavior and brain metabolism. In the prenatal human brain, vascular cells change metabolic states in the second trimester. Such findings highlight the need to identify new cellular and molecular players in neurodevelopmental disorders, raising awareness about the importance of a healthy vasculature for brain development. It is thus essential to shift the mostly neuronal point of view in research on ASD and other neurodevelopmental disorders to also include vascular and metabolic features.

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

BACKGROUND: Meningeal solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that are associated with local recurrence and hematogenous metastasis. The cell states and spatial transcriptomic architecture underlying the unique clinical behavior of meningeal SFTs are unknown. METHODS: Single-cell (n=4), spatial (n=8), and bulk RNA sequencing (n=22) was used to define the cell states and spatial transcriptomic architecture of meningeal SFTs across histological grades and in patient-matched pairs of primary/recurrent or intracranial/metastatic samples. Immunofluorescence, immunohistochemistry, and comparison of single-cell types to meningiomas, or to cerebral vascular development or homeostasis, were used for validation. RESULTS: Here we show meningeal SFTs are comprised of regionally distinct gene expression programs that resemble cerebral vascular development or homeostasis. Single-cell trajectory analysis and pseudotemporal ordering of single-cells suggest that meningeal SFT cell fate decisions are dynamic and interchangeable. Cell-cell communication analyses demonstrate receptor-ligand interactions throughout the meningeal SFT microenvironment, particularly between SFT cells, endothelia, and immature neurons. Direct comparison of single-cell transcriptomes from meningeal SFTs versus meningiomas shows that SFT cells are enriched in expression of endothelial markers while meningiomas cells are enriched in expression of mural cells markers. Meningeal SFT spatial transcriptomes show regionally distinct intratumor heterogeneity in cell states, gene expression programs, and cell-cell interactions across WHO histological grades and in patient-matched pairs of primary/recurrent or intracranial/metastatic samples. CONCLUSIONS: These results shed light on pathways underlying meningeal SFT biology in comparison to other central nervous system tumors and provide a framework for integrating single-cell, spatial, and bulk RNA sequencing data across human cancers and normal tissues.

Should neonatal-perinatal medicine move to two-year fellowships?

The duration of the majority of fellowships in pediatrics has been three-years. With increasing shortages of some outpatient-based pediatric subspecialists, shorter two-year fellowships are being considered for clinically oriented trainees not interested in a career based on research. Shortening the duration of fellowship may have some financial merits such as achieving a higher salary earlier after shorter training. However, we feel that continuing with a three-year duration for neonatology is more pragmatic at this time due to reductions in intensive care rotations during residency, time required to  achieve procedural excellence, the need for exposure to quality assurance methodology, proficiency in novel techniques such as bedside ultrasound, and to maintain the physician-scientist pipeline. The demand for neonatal fellowship continues to be high. Ongoing evaluation of the job market, training needs and fellowship curriculum is needed to determine if the duration of fellowship should be altered in the future.

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.

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 that 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. Significance: There are no effective systemic therapies to treat meningiomas, and meningioma stem cells are poorly understood. Here, we report perivascular NOTCH3+ stem cells to drive meningioma tumorigenesis and resistance to radiotherapy. Our results identify a conserved mechanism and a therapeutic vulnerability to treat meningiomas that are resistant to standard interventions.

Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity.

Glioblastoma (GBM) is the deadliest form of primary brain tumor with limited treatment options. Recent studies have profiled GBM tumor heterogeneity, revealing numerous axes of variation that explain the molecular and spatial features of the tumor. Here, we seek to bridge descriptive characterization of GBM cell type heterogeneity with the functional role of individual populations within the tumor. Our lens leverages a gene program-centric meta-atlas of published transcriptomic studies to identify commonalities between diverse tumors and cell types in order to decipher the mechanisms that drive them. This approach led to the discovery of a tumor-derived stem cell population with mixed vascular and neural stem cell features, termed a neurovascular progenitor (NVP). Following in situ validation and molecular characterization of NVP cells in GBM patient samples, we characterized their function in vivo. Genetic depletion of NVP cells resulted in altered tumor cell composition, fewer cycling cells, and extended survival, underscoring their critical functional role. Clonal analysis of primary patient tumors in a human organoid tumor transplantation system demonstrated that the NVP has dual potency, generating both neuronal and vascular tumor cells. Although NVP cells comprise a small fraction of the tumor, these clonal analyses demonstrated that they strongly contribute to the total number of cycling cells in the tumor and generate a defined subset of the whole tumor. This study represents a paradigm by which cell type-specific interrogation of tumor populations can be used to study functional heterogeneity and therapeutically targetable vulnerabilities of GBM.

Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain's innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.

Regulation of AID expression in the immune response.

The B cell-specific enzyme activation-induced cytidine deaminase (AID) has been shown to be essential for isotype switching and affinity maturation of antibody genes during the immune response. Conversely, AID activity has also been linked to autoimmunity and tumorigenesis. Determining how AID expression is regulated in vivo is therefore central to understanding its role in health and disease. Here we use phylogenetic footprinting and high-resolution histone acetylation mapping to accurately demarcate AID gene regulatory boundaries. Based on this strategy, we identify a novel, positive regulatory element required for AID transcription. Furthermore, we generate two AID indicator mouse strains using bacterial artificial chromosomes that faithfully recapitulate endogenous AID expression. The first strain uses a green fluorescent protein reporter to identify B cells that actively express AID during the immune response. In the second strain, AID transcription affects the permanent expression of a yellow fluorescent protein reporter in post-germinal center and terminally differentiated lymphocytes. We demonstrate the usefulness of these novel strains by resolving recent contradictory observations on AID expression during B cell ontogeny.

The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks.

DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.

Disentangling brain vasculature in neurogenesis and neurodegeneration using single-cell transcriptomics.

The vasculature is increasingly recognized to impact brain function in health and disease across the life span. During embryonic brain development, angiogenesis and neurogenesis are tightly coupled, coordinating the proliferation, differentiation, and migration of neural and glial progenitors. In the adult brain, neurovascular interactions continue to play essential roles in maintaining brain function and homeostasis. This review focuses on recent advances that leverage single-cell transcriptomics of vascular cells to uncover their subtypes, their organization and zonation in the embryonic and adult brain, and how dysfunction in neurovascular and gliovascular interactions contributes to the pathogenesis of neurodegenerative diseases. Finally, we highlight key challenges for future research in neurovascular biology.

Parents' Views on Autopsy, Organ Donation, and Research Donation After Neonatal Death.

Importance: Parents who experience neonatal loss have the option to participate in autopsy, organ donation, and research donation. However, clinicians are uncomfortable discussing autopsy and may not be aware of research and organ donation opportunities. Objective: To capture the perspectives of parents who had experienced neonatal loss about autopsy, organ donation, and research donation. Design, Setting, and Participants: This qualitative study used virtual focus groups with parents who attended a local bereavement support group in the US. Participants were recruited from Helping After Neonatal Death, a support group with a local chapter. Participants self-selected from an email request if they met the following criteria: aged 18 years or older, English speaking, at least 6 months elapsed since neonatal death, and access to a video conference device with internet. Focus groups took place between April and September 2021. The recorded sessions were analyzed using a grounded theory-informed approach by the research team that included parents with experience of neonatal loss. Data were analyzed from December 2021 through December 2022. Results: A total of 14 mothers engaged in the focus group; 9 (75%) were aged 30 to 39 years, and 8 (66%) were White. The mothers were overall well educated. The first main theme grew from the lived experience of neonatal loss, specifically the importance of offering all parents the option to donate, rather than prejudging who would or would not be interested. Parents of neonates who die have few opportunities to parent that child and make loving decisions for them. Participants emphasized that the conversation about autopsy, organ donation, and research donation, albeit difficult, can offer a meaningful parenting experience. A second main theme that emerged related to how organ or tissue donation could provide additional meaning to a child's life. These choices contributed to building a legacy to honor their child's memory, which also helped with grief and coping with their loss. A third theme included recommendations to clinicians and health systems for improving communication, including written information for parents and communication training for health care professionals. Conclusions and Relevance: In this qualitative study, parents who experienced neonatal loss endorsed the importance of offering parents the choice of autopsy, organ donation, or research donation with skillful and empathetic communication. They provided practical recommendations to improve communication and empower families.

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.

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.

Developmental dynamics of RNA translation in the human brain.

The precise regulation of gene expression is fundamental to neurodevelopment, plasticity and cognitive function. Although several studies have profiled transcription in the developing human brain, there is a gap in understanding of accompanying translational regulation. In this study, we performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human-specific and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem-cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and, thus, may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.

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.