Language is a defining trait of our species, and disruptions in language acquisition can have profound consequences to the individuals affected. Uncovering the neurodevelopmental basis of this complex trait requires detailed molecular and cellular insights into the neocortical areas that support linguistic abilities. Here we performed joint gene expression and chromatin accessibility profiling at single-nucleus resolution (10x Genomics Single cell Multiome) and spatial transcriptomic profiling (Xenium high-plex in situ spatial transcriptomics) of Broca's area alongside adjacent motor cortical areas. We profiled individuals from different ancestries (European and African) and developmental stages (infancy, childhood, adolescence, and adulthood). We provide a high-resolution dissection of the cellular and molecular architecture of Broca's and motor cortical areas across early life stages and anchor the trajectories to the cellular states found in the adult human brain. We identify distinct area- and stage-specific cellular signatures, including a prominent role of glia populations and interneuron subtypes contributing to cytoarchitectonic specializations. Using longitudinal single cell spatial transcriptomic profiling, we orthogonally validate our consensus cell taxonomy and spatially resolve layer enrichment of neuronal and astrocyte subtypes that distinguish Broca's area and motor cortex. We also uncover cell type-specific molecular signatures that distinguish cell developmental trajectories in these cortical areas, including an early molecular code established by differential expression of cadherin genes that might contribute to area-specific intercellular communication. We also identify cell type-specific vulnerabilities to language- related neurodevelopmental and neuropsychiatric disorders, with selective susceptibility of particular somatostatin-positive interneuron subtypes to ASD/ADHD. Finally, evolutionary analysis of differentially accessible regions between Broca's area and motor cortex suggests that genetic mutations that might have contributed to the emergence of linguistic abilities accumulated over the course of million years following the divergence of human and chimpanzee lineages. Together, our study provides a comprehensive molecular, cellular and spatial definition of Broca's area and motor cortex, laying the groundwork for investigations into unique aspects of human cognition and related neurodevelopmental and neuropsychiatric disorders.

Abstract Background: Whole genome sequencing (WGS) improves childhood cancer management by detecting clinically actionable variants beyond the scope of standard assays. However, current turnaround times (TAT) are well beyond clinical decision-making windows. We developed a novel ‘Ultra-Fast Whole Genome Sequencing’ (UF-WGS) technology to deliver comprehensive genomic results within days, and evaluated its feasibility, accuracy, and clinical impact in real-world pediatric hematology/oncology. Methods: Children with suspected or confirmed malignancy in Cambridge were recruited between 2023–2025. Tumor, bone marrow, and/or blood samples were analysed with UF-WGS and standard genomic medicine service (GMS-WGS) concurrently. UF-WGS deploys Constellation mapped read technology, eliminating library preparation by applying crude lysate or DNA directly onto the flowcell surface providing enhanced genome coverage, variant calling and phasing. Variant calls and TAT were benchmarked against GMS-WGS. Only clinically actionable variants as per the UK national genomics test directory were reported. Clinical utility was assessed by multidisciplinary review. Results: Fifty-four patients representing the expected range of childhood malignancies were recruited. UF-WGS reduced mean TAT of clinically actionable WGS reports from 37 to 3 days. It recalled 95% of somatic and germline variants and detected 19 additional variants, leading to alterations in clinical management. Discrepancies were due to tissue heterogeneity or low variant allele frequency. Median sequencing depth of UF-WGS (137× tumor; 84× germline) was superior to GMS-WGS (97x tumor; 42x germline).UF-WGS enabled demonstrable clinical benefit including risk stratification, target identification and pharmacogenomic guidance in 18/35 (51%) of prospectively recruited patients. UF-WGS supported de-escalation of therapy, earlier initiation of targeted treatments and optimization of surgical timing. For one patient, rapid identification of a germline ACVR1 mutation allowed a diagnosis of fibrodysplasia ossificans progressiva, avoiding harmful interventions. In 9/19 (47%) retrospective cases, independent reviewers judged that real-time UF-WGS would have improved management.UF-WGS was particularly advantageous in leukemia, where tumor-only analysis avoided delays associated with obtaining germline DNA. The clinical value of delivering comprehensive cytogenetic, minimal residual disease, and pharmacogenomic data within 72 hours is particularly great in this disease setting. Conclusions: UF-WGS is feasible, accurate and clinically impactful in pediatric cancer, delivering real-time WGS to inform management decisions. It offers a scalable framework to consolidate multiple molecular assays into a single, rapid test, supporting ambitions for faster genomic diagnosis and equitable precision medicine delivery internationally. Citation Format: Aditi Vedi, Jamie Trotman, Joao Dias, Martina Mijuskovic, Sera Choi, Laura Kingham, Rachel Moore, Sarah M. Leiter, Rowena Guermech, Amanda Semerene, Aviva Grisby, Sophie Wool, Victoria Joslin, Zoya Kingsbury, Mark Ross, David Bentley, Sam Behjati, Sean Humphray, Patrick Tarpey, David Rowitch. UltraFast whole genome sequencing enables personalized treatments in childhood cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3493.

The extent to which glial cell turnover features in successful remyelination is unclear. In this study, the rat caudal cerebellar peduncle-ethidium bromide lesion model was used to profile oligodendroglial and microglial/macrophage cell death and proliferation dynamics over the course of repair. Lesioned and control tissue was co-labelled with antibody markers for cell identity, proliferation, and apoptosis (TUNEL assay), then imaged at full thickness using confocal microscopy and quantified using custom CellProfiler pipelines. Early remyelination time points were marked by an increased density of total proliferating cells, including oligodendrocyte progenitor cells. Late remyelination time points featured increased TUNEL+ oligodendrocyte progenitor cells: however, most TUNEL+ cells within remyelinating lesions were Iba1+ microglia/macrophages. These results indicate that repairing lesions are characterized by a high degree of glial cell death and suggest that monitoring cell death-related by-products might have clinical value in the setting of remyelination. Gaitsch et al. used an experimental model of toxin-induced demyelination to profile glial cell death/proliferation dynamics over the course of remyelination. They report that significant oligodendrocyte progenitor and microglial cell death are features of remyelination. These results may guide future investigation of glial cell death by-products as remyelination biomarkers.

Whole genome sequencing (WGS) improves childhood cancer diagnosis, enabling precision treatment. However, timely clinical decisions are made within days, while WGS clinical reports using current technologies and workflows, can exceed 6 weeks. Here we report an ‘Ultra-fast WGS (UF-WGS)’ workflow, providing rapid and acurate molecular profiling of childhood malignancies. In 54 children with suspected or confirmed cancer, UF-WGS reduced turnaround time from 37 to 3 days. It captured 95% of clinically actionable variants found by conventional methods, and identified 19 additional actionable variants. UF-WGS led to demonstrable improvements in care for 51% of prospective patients, including avoidance of over-medicalisation and support for timely precision therapy. UF-WGS was feasible across diverse tumour types and sample sources, with additional technical advantages in variant detection and workflow simplicity. These findings indicate UF-WGS can significantly improve the management of paediatric haematology/oncology patients, specifically through precision diagnosis, risk stratification and molecularly informed treatment escalation/de-escalation.

Genetic variants linked to autism are thought to change cognition and behaviour by altering the structure and function of the brain. Although a substantial body of literature has identified structural brain differences in autism, it is unknown whether autism-associated common genetic variants are linked to changes in cortical macro- and micro-structure. We investigated this using neuroimaging and genetic data from adults (UK Biobank, N = 31,748) and children (ABCD, N = 4928). Using polygenic scores and genetic correlations we observe a robust negative association between common variants for autism and a magnetic resonance imaging derived phenotype for neurite density (intracellular volume fraction) in the general population. This result is consistent across both children and adults, in both the cortex and in white matter tracts, and confirmed using polygenic scores and genetic correlations. There were no sex differences in this association. Mendelian randomisation analyses provide no evidence for a causal relationship between autism and intracellular volume fraction, although this should be revisited using better powered instruments. Overall, this study provides evidence for shared common variant genetics between autism and cortical neurite density.

Background and ObjectivesHypotonia is a relatively common finding among infants in the neonatal intensive care unit (NICU). Consideration of genetic testing is recommended early in the care of infants with unexplained hypotonia. We aimed to assess the diagnostic yield and overall impact of exome and genome sequencing (ES and GS).MethodsConsecutive infants with hypotonia were identified from research and clinical databases across 5 teaching hospitals in United States, Canada, United Kingdom, and Australia. Inclusion criteria included NICU admission and genetic evaluation. Infants with a known explanation for hypotonia were excluded. Data regarding infant characteristics, genetic testing, and diagnoses were collected. The primary outcome was identification of a molecular diagnosis. Impact on care was a secondary outcome. The Fisher exact and Wilcoxon rank-sum tests were used for statistical analysis.ResultsWe identified 147 infants with unexplained hypotonia. The median gestational age was 39 weeks (interquartile range [IQR] 36-42 weeks), 77 (52%) were female, and the median age was 8 days at the time of evaluation (IQR 2-19 days). Eighty (54%) had hypotonia as the main clinical feature while 67 (46%) had additional multisystem involvement. Seventy-five (51%) underwent rapid ES, 44 (30%) rapid GS, 2 (1%) both ES and GS, and 26 (18%) were admitted before ES or GS became available. Of the 121 infants who underwent ES and/or GS, 72 (60%) had the primary outcome of a molecular diagnosis. In addition, 2 infants with mitochondrial genome variants were diagnosed by mitochondrial GS after negative ES, and one infant needed targeted testing to identify a short tandem repeat expansion missed by GS. The proportion diagnosed by ES and GS was not different between infants with hypotonia as the primary finding (37/56, 66%) and infants with multisystemic symptoms (35/65, 54%, odds ratio [OR] 1.7, CI 0.8-3.7, p value = 0.20). Testing was more likely to have an impact on care for infants receiving a genetic diagnosis (57/66 vs 14/33, OR 8.4, CI 2.9-26.1, p = 1.0E-05).DiscussionRapid ES and GS provided a molecular diagnosis for most of the infants with unexplained hypotonia who underwent testing. Further studies are needed to assess the generalizability of these findings as increased access to genetic testing becomes available.Classification of EvidenceThis study provides Class IV evidence that in unexplained neonatal hypotonia, rapid ES or GS adds diagnostic specificity.

Background: Whole Genome Sequencing (WGS) enhances paediatric cancer diagnosis and management compared with standard molecular assays. However, its clinical utility could be further improved by reducing the National Health Service England (NHSE) turnaround times (TAT). Methods: We evaluated an Ultra-Fast WGS (UF-WGS) workflow in a tertiary UK paediatric haematology-oncology unit. Children with suspected or confirmed cancer were recruited over two years (2023-2025), and their tumour, bone marrow and/or blood samples were sequenced on the UF-WGS workflow. All patients underwent concurrent NHSE Genomic Medicine Service (GMS) WGS, serving as the validation benchmark. Results: Of a total of 54 patients were recruited at diagnosis or relapse. UF-WGS reduced TAT to a mean of 3 days from sample collection, compared with 37 days for GMS-WGS. UF-WGS recalled 95% (143/151) of all clinically actionable somatic and germline variants not found standard NHS GMS-WGS testing. UF-WGS detected an additional 19 clinically actionable variants not found by GMS-WGS. Differences between the two workflows were attributable to tumour heterogeneity in some cases, and low variant allele frequency of those variants identified discrepantly. Additionally, in 18/35 (51%) prospective cases, UF-WGS enabled demonstrable improvements in care. Clinicians independently judged that 9/19 (47%) of the retrospective cases would have clinically benefited from real-time UF-WGS. UF-WGS provided additional flowcell proximity data, which illustrated the potential to positively impact clinical care. Conclusions: This study indicates the feasibility and utility of UF-WGS and shows added benefits for the clinical management of paediatric cancers, with wider implications beyond this patient group. ### Competing Interest Statement MM, SC, PG, ZK, JB, TN, IA, IV, MB, LF, JW, MR, DB and SH are or were employees of Illumina at the time of the study, a public company that develops and markets systems for genetic analysis. ### Clinical Trial 22/WA/0336 ### Clinical Protocols ### Funding Statement This work was supported by the Rosetrees Trust (AV, DHR), Addenbrookes Charitable Trust (AV), Isaac Newton Trust (DHR) and the NIHR Cambridge Biomedical Research Centre (NIHR203312). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Full ethical approval granted by the Health Research Authority and Health Care Research Wales (REC reference 22/WA/0336). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript, and supplementary tables. Any additional data may be available upon reasonable request to the authors

Glioma stem cells (GSCs) from this aggressive brain cancer have been subject to nononcogene addiction therapeutic strategies, in particular targeting iron and cholesterol metabolic pathways. In this study, we show the small molecule Adaptaquin (AQ) has anti‐GSC effects while sparing neurons, mature oligodendrocytes and astrocytes. Transcriptomic analysis of AQ‐treated GSCs showed dramatic upregulation of iron transport genes and downregulation of genes involved in cholesterol biosynthesis. Indeed, we found cytotoxic effects of AQ on GSCs were potentiated when combined with the iron chelator deferoxamine (DFO). Notably, these effects were independent of PHD2 and HIF1α regulation, indicating a distinct pathway of action. Furthermore, we observed that the heme analogue, hemin, protects GSCs from AQ‐mediated cell death, suggesting the presence of a functional heme transporter in GSCs, an observation confirmed by uptake of heme analogues. Importantly, we found that AQ treatment alone or in combination with iron chelators impaired cholesterol homeostasis in GSCs, leading to mitochondrial fragmentation and cell death. These findings suggest AQ in combination with iron chelators results in lethal disruption of cholesterol metabolism in glioma stem cells.

Cancer cells display highly heterogeneous and plastic states in glioblastoma, an incurable brain tumour. However, how these malignant states arise and whether they follow defined cellular trajectories across tumours is poorly understood. Here, we generated a deep single cell and spatial multi-omic atlas of human glioblastoma that pairs transcriptomic, epigenomic and genomic profiling of 12 tumours across multiple regions. We identify that glioblastoma heterogeneity is driven by spatially-patterned transitions of cancer cells from developmental-like states towards those defined by a glial injury response and hypoxia. This cellular trajectory regionalises tumours into distinct tissue niches and manifests in a molecularly conserved manner across tumours as well as genetically distinct tumour subclones. Moreover, using a new deep learning framework to map cancer cell states jointly with clones in situ , we show that tumour subclones are finely spatially intermixed through glioblastoma tissue niches. Finally, we show that this cancer cell trajectory is intimately linked to myeloid heterogeneity and unfolds across regionalised myeloid signalling environments. Our findings define a stereotyped trajectory of cancer cells in glioblastoma and unify glioblastoma tumour heterogeneity into a tractable cellular and tissue framework. ### Competing Interest Statement J.S.R. reports funding from GSK, Pfizer and Sanofi & fees/honoraria from Travere Therapeutics, Stadapharm, Astex, Owkin, Pfizer, Grunenthal, Moderna and Tempus. O.S. is a paid advisor of Insitro. The other authors declare no competing interests. Wellcome Leap as part of the Delta Tissue

Cortical GABAergic interneurons generated in the ventral developing brain travel long distances to their final destinations. While there are examples of interneuron migration in the neonatal human brain, the extent of postnatal migration across species and how it contributes to cortical interneuron composition remains unknown. Here we demonstrate that neonatal gyrencephalic brains, including humans, nonhuman primates and piglets, harbor an elaborate subventricular zone, termed the Arc, due to its curved morphology and expanded neuroblast populations. The Arc is absent in lissencephalic marmoset and mouse brains. Transcriptomic and histological approaches revealed that Arc neurons are diverse interneurons from the medial and caudal ganglionic eminences that migrate into the frontal, cingulate and temporal cortex. Arc-cortical targets exhibit an increase in VIP+ neuronal density compared to other regions. Our findings reveal that the Arc is a developmental structure that supports the expansion of postnatal neuronal migration for cortical interneuron patterning in gyrencephalic brains.

Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.

The importance of neuroinflammation in neurodegenerative diseases is becoming increasingly evident, and, in parallel, human induced pluripotent stem cell (hiPSC) models of physiology and pathology are emerging. Here, we review new advancements in the differentiation of hiPSCs into glial, neural, and blood–brain barrier (BBB) cell types, and the integration of these cells into complex organoids and chimeras. These advancements are relevant for modeling neuroinflammation in the context of prevalent neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS). With awareness of current limitations, recent progress in the development and application of various hiPSC-derived models shows potential for aiding the identification of candidate therapeutic targets and immunotherapy approaches.

BACKGROUND AND OBJECTIVES:Hypotonia is a relatively common finding among infants in the neonatal intensive care unit (NICU). Consideration of genetic testing is recommended early in the care of infants with unexplained hypotonia. We aimed to assess the diagnostic yield and overall impact of exome and genome sequencing (ES and GS). METHODS:Consecutive infants with hypotonia were identified from research and clinical databases across 5 teaching hospitals in United States, Canada, United Kingdom, and Australia. Inclusion criteria included NICU admission and genetic evaluation. Infants with a known explanation for hypotonia were excluded. Data regarding infant characteristics, genetic testing, and diagnoses were collected. The primary outcome was identification of a molecular diagnosis. Impact on care was a secondary outcome. The Fisher exact and Wilcoxon rank-sum tests were used for statistical analysis. RESULTS:We identified 147 infants with unexplained hypotonia. The median gestational age was 39 weeks (interquartile range [IQR] 36-42 weeks), 77 (52%) were female, and the median age was 8 days at the time of evaluation (IQR 2-19 days). Eighty (54%) had hypotonia as the main clinical feature while 67 (46%) had additional multisystem involvement. Seventy-five (51%) underwent rapid ES, 44 (30%) rapid GS, 2 (1%) both ES and GS, and 26 (18%) were admitted before ES or GS became available. Of the 121 infants who underwent ES and/or GS, 72 (60%) had the primary outcome of a molecular diagnosis. In addition, 2 infants with mitochondrial genome variants were diagnosed by mitochondrial GS after negative ES, and one infant needed targeted testing to identify a short tandem repeat expansion missed by GS. The proportion diagnosed by ES and GS was not different between infants with hypotonia as the primary finding (37/56, 66%) and infants with multisystemic symptoms (35/65, 54%, odds ratio [OR] 1.7, CI 0.8-3.7, p value = 0.20). Testing was more likely to have an impact on care for infants receiving a genetic diagnosis (57/66 vs 14/33, OR 8.4, CI 2.9-26.1, p = 1.0E-05). DISCUSSION:Rapid ES and GS provided a molecular diagnosis for most of the infants with unexplained hypotonia who underwent testing. Further studies are needed to assess the generalizability of these findings as increased access to genetic testing becomes available. CLASSIFICATION OF EVIDENCE:This study provides Class IV evidence that in unexplained neonatal hypotonia, rapid ES or GS adds diagnostic specificity.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function. Here the authors show that ventrally derived oligodendrocytes (OLs) can myelinate areas usually populated by dorsally derived OLs but cannot functionally compensate, as animals populated only by ventrally derived OLs show locomotor and cognitive deficits.

The role of reactive astrocytes in perinatal white matter injury (WMI) is unclear. In a mouse model of WMI, we provide evidence that impairing the formation of a C3 -expressing neuroinflammatory reactive astrocyte sub-state rescues myelination and behavioral deficits. We further demonstrate the presence of C3 -expressing reactive astrocytes in human WMI. Our data point to these cells as putative drivers of myelination failure in WMI and a potentially promising therapeutic target.

Oligodendrocytes (OLs) of the central nervous system require iron for proteolipid biosynthesis during the myelination process. Although most heme is found complexed to hemoglobin in red blood cells, surprisingly, we found that Slc48a1, encoding the heme transporter Hrg1, is expressed at higher levels in OLs than any other cell type in rodent and humans. We confirmed in situ that Hrg1 is expressed in OLs but not their precursors (OPCs) and found that Hrg1 proteins in CNS white matter co-localized within myelin sheaths. In older Hrg1 null mutant mice we observed reduced expression of myelin associated glycoprotein (Mag) and ultrastructural myelin defects reminiscent of Mag-null animals, suggesting myelin adhesion deficiency. Further, we confirmed reduced myelin iron levels in Hrg1 null animals in vivo, and show that OLs in vitro can directly import both the fluorescent heme analogue ZnMP and heme itself, which rescued iron deficiency induced inhibition of OL differentiation in a heme-oxidase-dependent manner. Together these findings indicate OL Hrg1 encodes a functional heme transporter required for myelin integrity.

The original description of cerebral palsy (CP) contained case histories suggesting that perinatal environmental stressors resulted in brain injury and neurodevelopmental disability. While there are clear associations between environmental impact on brain development and CP, recent studies indicate an 11% to 40% incidence of monogenic conditions in patients given a diagnosis of CP. A genetic diagnosis supports the delivery of personalized medicine. In this review, we describe how the Wnt pathway exemplifies our understanding of pathophysiology related to a gene variant (CTNNB1) found in some children diagnosed with CP. We cover studies undertaken to establish the baseline prevalence of monogenic conditions in populations attending CP clinics. We list factors indicating increased likelihood of a genomic diagnosis; and we highlight the need for a comprehensive, accurate, genotype-phenotype reference data set to aid variant interpretation in CP cohorts. We also consider the wider societal implications of genomic management of CP including significance of the diagnostic label, benefits and pitfalls of a genetic diagnosis, logistics, and cost.

Abstract AIMS Glioblastoma (GBM) recurrence is rooted in the ability of residual stem cells to survive treatment and drive relapse. One way to eradicate glioblastoma stem cells (GSCs) is to differentiate them into post-mitotic, non-tumorigenic cell types. Here we aim to differentiate GSCs via genetic manipulation, and test how their response differs in 2D and 3D cultures. METHOD We generated a co-culture model to grow GSC lines with iPSC-derived 3D human brain organoids. Before co-culture, the GSC lines were genetically engineered to overexpress a Doxycycline (Dox) inducible form of the proneural transcription factor ASCL1 (wild type and phosphorylation defective). Live imaging and immunochemistry were performed to analyse tumor composition. RESULTS Our data show that activation of the proneural factor ASCL1 in GSCs when co-cultured with 3D human brain organoids drives reduction in tumor growth and that inhibition of ASCL1 phosphorylation only slightly improves this effect. In comparison to data in 2D cultures, the effect of ASCL1 activation seems enhanced in 3D organoids. At day 7 and 14 of ASCL1 induction, there are only few cancer cells left in the organoids, while, in 2D, wildtype ASCL1-expressing cells continue to grow, even if at a slower rate than control GSCs. CONCLUSION Our data show that cancer stem cells embedded in 3D human brain organoids behave differently than when in 2D culture on plastic and respond more effectively to differentiation treatments. Our model provides a platform to test novel treatments for GBM.

Perinatal white matter injury (WMI) is the leading cause of long-term neurological morbidity in infants born preterm. Neuroinflammation during a critical window of early brain development plays a key role in WMI disease pathogenesis. The mechanisms linking inflammation with the long-term myelination failure that characterizes WMI, however, remain unknown. Here, we investigate the role of astrocyte reactivity in WMI. In an experimental mouse model of WMI, we demonstrate that WMI disease outcomes are improved in mutant mice lacking secretion of inflammatory molecules TNF-α, IL-1α, and C1q known, in addition to other roles, to induce the formation of a neuroinflammatory reactive astrocyte substate. We show that astrocytes express molecular signatures of the neuroinflammatory reactive astrocyte substate in both our WMI mouse model and human tissue affected by WMI, and that this gene expression pattern is dampened in injured mutant mice. Our data provide evidence that a neuroinflammatory reactive astrocyte substate correlates with adverse WMI disease outcomes, thus highlighting the need for further investigation of these cells as potential causal players in WMI pathology.