Cell Reports Methods

Cortical organoids (COs) represent a powerful in vitro model system that recapitulates key aspects of human brain development, enabling the study of neurodevelopmental processes, cellular diversity, and disease mechanisms in a physiologically relevant 3D environment. However, traditional histological analysis of COs relies on tissue sectioning, which limits the ability to capture the full spatial complexity of organoid architecture. In this study, we establish a framework for applying CLARI-O, an improved tissue-clearing technique, for intact COs and organoid-based systems, enabling comprehensive 3D visualization and analysis of 3D organizational features. Using CLARI-O in combination with high-resolution imaging, we demonstrate the utility of tissue clearing for studying glial populations, including oligodendrocytes and microglia, considered to be underrepresented in COs, and their interactions with neurons. Additionally, we apply this method to forebrain assembloids (FAs) to visualize cellular heterogeneity and the interface between ventral and dorsal regions. Finally, we use CLARI-O to study mouse brains containing xenotransplanted COs (MB-COs) to evaluate human cell integration, migration, vascularization, and structural connectivity. This is the first study to demonstrate how tissue clearing can be used after functional assays such as calcium imaging to correlate neural activity with post hoc structural analysis in MB-COs. Together, this work establishes CLARI-O as a powerful tool for advancing 3D structural and functional interrogation of human CO-derived systems, enhancing their value for disease modeling, drug screening, and translational neuroscience. MotivationCortical organoids have become an increasingly powerful tool in neuroscience. Their complexity has expanded substantially, now incorporating exogenous lineages, fusing organoids with distinct regional identities (assembloids), and enabling xenotransplantation into in-vivo environments. These advancements require more sophisticated technological approaches that are capable of capturing the intricate three-dimensional cyotarchitecture and organization of intact organoid systems both in vitro and after xenotransplantation in vivo. Tissue-clearing methodologies offer a unique opportunity to visualize these structural and cellular features with exceptional depth and resolution. Graphical abstract HighlightsO_LIWe optimized clearing protocols to develop an organoid specific clearing method (CLARI-O) that enables high-resolution visualization of diverse neuronal and glial populations without tissue sectioning, preserving long-range connections and cellular processes. C_LIO_LIForebrain assembloids used to study neuronal and oligodendrocyte migration can be effectively processed using CLARI-O, allowing detailed visualization of fusion interface. C_LIO_LIWe established a robust framework for CLARI-O-based clearing of mouse brain tissue containing xenotransplanted human cortical organoids, enabling comprehensive 3D analysis of graft development, integration, and vascularization in vivo. C_LI

Studying neural-related questions is inherently challenging due to the limited number of suitable cell models. Here, we characterize a previously reported immortalized human neural stem cell line, HNSC.100, serving as a robust model for a wide range of neurobiological research questions. The cell line expresses key neural stem cell markers, including SOX2, vimentin, nestin, and allows for efficient genetic manipulation. Furthermore, HNSC.100 cells can be differentiated into neurons, astrocytes and oligodendrocytes, thereby covering a wide spectrum of major neural cell types. We established a comprehensive panel of molecular markers to validate successful differentiation, enabling precise characterization of the resulting cell population. In addition, we provide a complete dataset of RNA expression levels for all detectable genes in HSNC.100 cells. Based on this dataset, we assembled a list of expressed genes implicated in neural disorders that can be studied with this cell line. Together, we present a detailed characterization of the HNSC.100 cell line and provide new tools and reference data to facilitate its use. This resource enables researchers to evaluate the lines suitability for specific applications and to rapidly integrate HNSC.100 cells into their experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/700829v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@115f8b3org.highwire.dtl.DTLVardef@17adb69org.highwire.dtl.DTLVardef@dad583org.highwire.dtl.DTLVardef@f7a691_HPS_FORMAT_FIGEXP M_FIG C_FIG

Synapses are the basic unit of information transfer between neurons. Their dysfunction is a common trigger of cognitive diseases and disorders. However, high-throughput analysis methods to assess synaptic function and dysfunction are lacking. Calcium imaging in cultured neurons in the absence of Mg2+ and presence of TTX allows visualization of NMDAR-dependent spontaneous synaptic calcium transients, which report pre and postsynaptic function. Here, we introduce a high-throughput automated analysis pipeline that combines Suite2p ROI detection and Python scripts to analyze tens of thousands of synapses and quantify changes in presynaptic vesicle fusion rates (frequency), postsynaptic function (amplitude), and the number of functional synapses. We use this pipeline to test known NMDAR agonists (glycine) and antagonists (ketamine, memantine, APV), presynaptic function modulating compounds (PDBu), and encephalitis patient-derived NMDAR auto-antibodies, where our pipeline proved more sensitive in detecting dysfunction at the single-synapse level than other methods. The ability to detect, track, and quantify activity across tens of thousands of synapses and millions of synaptic calcium transients using this pipeline will aid drug discovery of compounds that protect synapse function.

MOTIVATIONLongitudinal molecular studies of the mouse brain are limited by the need for terminal tissue collection. This prevents analysis of preexisting molecular states and their evolution within the same individual. We developed a stereotactic microbiopsy technique that enables minimally invasive sampling of defined brain regions in vivo. The method preserves survival while yielding material suitable for RNA and nuclei isolation. It provides a practical solution for linking baseline molecular states to subsequent behavioural, pharmacological, or disease-related outcomes. SUMMARYThis study presents a stereotactic microbiopsy technique for sampling defined brain regions in living mice, enabling transcriptomic and epigenomic analyses without sacrificing the animal. The method will allow pre-intervention tissue collection, making it possible to separate preexisting molecular differences from experience- or treatment-induced changes. We show that microbiopsies yield sufficient, high-quality RNA and chromatin for sequencing, with minimal tissue damage that largely resolves over time. The procedure uses standard stereotactic equipment and achieves reproducible spatial precision when the syringe is stabilised. This approach provides a practical framework for within-subject molecular comparisons, reducing animal use and enabling longitudinal profiling of the living mouse brain. It establishes a foundation for investigating how baseline molecular states influence later physiological or behavioural outcomes.

Dynamics of messenger ribonucleic acids (mRNAs) and the complexes with associated proteins (RNPs), also known as RNA granules, provide post-transcriptional control of gene expression. This regulation is crucial for cell-type diversity and activity-dependent plasticity of the mammalian central nervous system (CNS). However, elucidating the physiological significance of RNA granules has been hampered by the lack of technologies for probing them in live mouse brain. Here, we describe a novel method to visualize RNA granules in native CNS tissues in vivo. To amplify the fluorescence signal from single mRNAs incorporating MS2 stem loops, MS2 capsid protein (MCP) was conjugated with 4 tandem superfolder green fluorescent proteins (sfGFPs). Using MCP-4xsfGFP, a significant population of RNPs could be detected in specific cells of the CNS, enabling new findings, e.g., remarkable heterogeneity of Actb mRNA dynamics across neurons and glial cells. The highly sensitive in vivo RNA imaging could be useful for illuminating the regulation of the dynamics of RNA granules in naive and diseased animals. SignificanceRNA granules, complexes of RNAs and binding proteins, play a vital role in regulating gene expression and mRNA dynamics. Despite the importance, monitoring their behavior in functional neural tissue has proven technically challenging, in part due to optical turbidity and weak fluorescent tags. In this study, we present a new approach to better observe RNA granules in specific cells of murine brain through the use of amplified fluorescent signals and advanced imaging techniques. Our method provides a powerful means to analyze the properties of RNA granules within neurons and glial cells in vivo, offering valuable insights into healthy and neurodegenerative brains.

Parvalbumin-expressing cortical interneurons play a critical role in maintaining the balance between excitatory and inhibitory signalling and are essential for cognition, with dysfunction implicated in numerous brain disorders. Although human pluripotent stem cells have enabled the generation of diverse human neuronal types in vitro, including cortical interneurons, parvalbumin-expressing interneurons - unlike somatostatin-expressing interneurons - remain difficult to generate reliably and consistently. Here, we demonstrate the efficient and reproducible generation of parvalbumin-expressing cortical interneurons in vitro within 50 days of differentiation. Parvalbumin mRNA and protein were detected without forced gene expression, cell sorting, rodent co-culture or intracerebral transplantation, approaches commonly required by previous protocols. Single-cell transcriptomic analyses validated neuronal identity and authenticity, revealing enrichment for gene expression signatures of parvalbumin-expressing cortical interneurons in vivo. Together, these findings establish a robust method that facilitates interneuron research by enabling the reliable generation of authentic human parvalbumin-expressing cortical interneurons within a short time frame. eTOC blurbAzzouni et al. present a rapid and reproducible protocol for generating authentic human parvalbumin-expressing cortical interneurons from pluripotent stem cells in just 50 days, without forced gene expression or co-culture. Single-cell transcriptomics confirm robust acquisition of in vivo-like PVALB interneuron identity, enabling new opportunities for human interneuron research. HighlightO_LIOptimising SHH and WNT modulators enables consistent PVALB interneuron generation. C_LIO_LI10% of cells express PVALB mRNA within 50 days of 2D differentiation from hPSCs. C_LIO_LIPVALB expression occurs without gene forcing, sorting, co-culture or grafting. C_LIO_LIComparison of gene expression to in vivo interneurons confirms PVALB authenticity. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/710579v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a53d13org.highwire.dtl.DTLVardef@14cd131org.highwire.dtl.DTLVardef@3a0f9corg.highwire.dtl.DTLVardef@1d7034d_HPS_FORMAT_FIGEXP M_FIG C_FIG

APOBEC-mediated cytidine deamination is a major endogenous source of mutagenesis in human cancers and has been linked to tumor evolution, clonal diversification and therapeutic resistance. Among the APOBEC family, APOBEC3A (A3A) is a potent and inducible cytidine deaminase, with dynamic and context-dependent activation. Most approaches for studying the role of A3A in cancer infer A3A activity indirectly via its expression level or retrospective mutational signatures, or through molecular assays that are limited to endpoint measurements and do not readily allow longitudinal interrogation of A3A editing dynamics. Therefore, quantifying the timing, persistence, and cellular heterogeneity of A3A activity remains challenging. Here, we describe ApoFLARE, a genetically encoded reporter that converts A3A-mediated cytidine deamination into a quantitative luminescent signal in living cells. ApoFLARE allows for scalable, ratiometric measurement of editing activity and enables time-resolved analysis of editing kinetics. Reporter activation is selectively dependent on A3A catalytic function and was absent in A3A-deficient, but not A3B-deficient cells. Under stress and targeted therapy conditions, reporter activity correlated with endogenous RNA editing measured by digital droplet PCR, including contexts in which catalytic activity persisted beyond transient A3A transcript induction. Thus, ApoFLARE offers a scalable platform to investigate the regulation, kinetics, and heterogeneity of A3A editing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/710312v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19497aborg.highwire.dtl.DTLVardef@71866borg.highwire.dtl.DTLVardef@12ffbdaorg.highwire.dtl.DTLVardef@13fd501_HPS_FORMAT_FIGEXP M_FIG C_FIG

To improve our understanding of synapse assembly, there is a need for robust, easy-to-use, and physiologically relevant in-vitro models allowing the controllable formation of neuronal contacts in a reasonable time, whose structure and function can be investigated using advanced microscopy. To address this challenge, we engineered 3D cultures from rodent dissociated hippocampal cells, that spontaneously assemble in low attachment U-bottom wells into compact spheroids of reproducible dimensions (100-300 microns), determined by the number of seeded cells. These neurospheres contain a mix of neurons and glial cells and grow over time in culture, through the combination of cell proliferation and neurite extension. Neurospheres were immunostained in fluid phase, and/or sparsely electroporated for the multi-color visualization of synaptic proteins. Neurons extend an elaborate network of axons and dendrites, forming within 2 weeks numerous excitatory and inhibitory synapses identified at the structural level by confocal and electron microscopy, and at the functional level by electrophysiology. Periodic calcium oscillations throughout neurospheres further highlight network activity. Finally, we demonstrate the potential of neurospheres to study synaptogenesis by modulating and visualizing the adhesion protein neuroligin-1. Overall, neurospheres represent a standardized and cost-effective system to study synapse structure and function at high resolution in 3D, that should be quite appealing to the cellular neurobiology community.

Metabolic dysregulation is increasingly recognized as a key contributor to neurodevelopmental disorders. Here, we present Intelliwaste, a non-invasive, cost-effective method for profiling carbon metabolism in pluripotent stem cells and brain organoids using 13C-labeled metabolites and 1H and 13C NMR spectroscopy. This approach enables longitudinal analysis of extracellular fluxes without disrupting cell viability. We apply Intelliwaste to human embryonic stem cells (hESCs) cultured in a defined media enriched with >95% 13C1-Glucose. Under these conditions, 13C3-lactate emerged as the most abundant labeled product, with 20-50-fold lower fluxes to 13C3-alanine, 13C2-acetate, 13C3-serine, and 13C3-pyruvate, and 100-300-fold lower fluxes to 13C1-formate and multiple 13C-labeled glutamate species. These profiles allow for precise quantification of fractional metabolic isotopic labeling and glucose-derived carbon flow. To demonstrate biological utility, we first examine the effect of L-glutamine omission, which selectively reduces 13C3-alanine/13C3-lactate and 13C4-glutamate/13C3-lactate flux ratios, while the 13C3-Glutamate/13C3-Lactate and 13C2-Glutamate/13C3-Lactate flux ratios remained unchanged. These findings suggest a specific role for extracellular glutamine in modulating the activity of alanine aminotransferase and pyruvate carboxylase. We then characterized LIS1 mutant hESCs--a model of lissencephaly--and observed significantly increased flux ratios involving 13C4-, 13C3-, and 13C2-glutamate relative to 13C3-lactate, indicating enhanced glutamate production via the TCA cycle. These findings establish Intelliwaste as a powerful tool for metabolic profiling in the study of human neurodevelopment and disease. Its non-destructive nature makes it particularly well-suited for tracking metabolic changes during differentiation and in patient-derived organoid models of neurological disorders.

Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells (HSCs) represents a promising approach to advance cell and gene therapy strategies yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by single cell RNA sequencing, which highlighted differentiation trajectories with preservation of lineage fidelity in committed progenitors. Defining a putative HSC cluster allowed an estimation of transduction efficiency in ex vivo cultures, which correlated with long-term gene marking in xenografts and patients enrolled in a gene therapy study. We then developed a clinically translatable, GMP-compliant process to expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult donors, by biologically informed protocol improvements of cytokine supplementation, media choice, timing of LV transduction and combinations of small molecules preventing the activation of differentiation programs. Our optimized process outperforms validated state-of-the-art cord blood expansion protocols when applied to mPB. LV integration site analysis and genomic barcode-based clonal tracking provided definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo culture. These results warrant clinical testing of this HSC transduction/expansion process in an upcoming clinical gene therapy trial for autosomal recessive osteopetrosis (EU CT 2024-518972-30). One Sentence SummaryA mobilized peripheral blood HSC expansion protocol optimized for gene therapy allows robust polyclonal long-term engraftment of LV-transduced cells.

The establishment of genetic circuits in pluripotent stem cells (PSCs) allows to model and manipulate developmental events. However, prototyping complex circuitry remains challenging, due to limitations in screening circuit components and transgene silencing. Here, we introduce KELPE: PSCs with two silencing-resistant insulated genomic landing pads targeted to genomic safe harbour sites. KELPE cells enable the stable integration of multiple transgenes into the same genomic region, facilitating fair comparisons of genetic circuit components. We demonstrate this by fine-tuning "synthetic neighbour-labelling" technologies. We first generate optimised PUFFFIN PSCs, which report on cell-cell interactions by fluorescently labelling wild-type neighbours. We then generate new synNotch "receiver" PSCs, which can trigger expression of any transgene following interaction with a synthetic ligand presented by "sender" cells of interest. We describe an optimised circuit syntax that abolishes ligand-independent transgene induction in receiver PSCs, and showcase this by synthetically programming cell death in receiver cells engineered to express a toxin following interaction with sender cells. In summary, we describe a new cell line that facilitates silencing-resistant transgene expression and prototyping of synthetic biology tools in a developmentally-relevant model.

Facial development is highly sensitive to genetic and environmental perturbation, with craniofacial malformation associated with over one-third of congenital birth defects. The face arises during an early and largely inaccessible window of embryonic development, with a large contribution from transient and multipotent cranial neural crest cells (CNCCs). Assessment of the molecular and cellular mechanisms driving normal and disordered human facial development therefore relies greatly on the use of in vitro cellular models. Here, we adapted a neurosphere-based CNCC differentiation protocol to facilitate robust quantification of early specification and migration events. Introduction of single-cell aggregation with arrayed plating enabled standardisation of neurosphere size, growth and patterning. Inclusion of fibronectin coating enhanced the efficiency of neurosphere attachment and synchronicity of CNCC migration timing. To demonstrate application of the Array-CNCC method, we developed a strategy for mosaic co-culture, which can facilitate differentiation of wildtype untreated cells directly alongside cells exposed to distinct drug treatments or genetic alterations. Finally, we present a screening approach which we use to test the impact of distinct extracellular matrix components on neurosphere morphology, CNCC migration and gene expression. Together, the Array-CNCC method is highly amenable to quantitative phenotyping and screening approaches, enabling enhanced craniofacial disease modelling with both cellular and molecular readouts.

Cellular therapies, toxicity screening, and regenerative medicine depend on selecting mammalian cell types with optimal lifespan, persistence post-transplant, immunogenicity, and chemical resilience. This review synthesizes data from over 50 immune, parenchymal, stem, and emerging engineered cell populations--including gamma-delta T cells, iNKT cells, CAR-macrophages, and hypoimmune iPSC derivatives--drawing from in vivo lifespan studies (including 1{blacksquare}C birth-dating and deuterium labeling), engraftment dynamics, immune rejection risk, and stress sensitivity profiles. We introduce a Programmability & Persistence Score (PPS; 0-20) that integrates these features into a unified metric, complemented by Pareto frontier analysis to visualize multi-objective trade-offs. High-PPS cell types (e.g., HLA-matched HSCs, hypoimmune iPSCs, chondrocytes) are suited for long-term regenerative applications, while low-PPS sentinels (e.g., neutrophils, enterocytes) serve acute assays. We discuss mathematical extensions including multi-criteria decision analysis, fuzzy membership functions, and Bayesian frameworks that address limitations of linear additive scoring. Together, these integrated profiles support cell selection for gene editing, organ-on-chip systems, in vivo cell programming, and immunotherapy, bridging cell biology with translational engineering.

Antigen-specific T cell populations are of great value for studying immune recognition but tedious to generate by limiting dilution or cloning. Here, we develop a streamlined approach to generate antigen-specific T cell clones directly from peripheral blood using the cloneXplorer, a live-cell analysis and clone isolation platform based on conical microwell arrays. This platform continuously monitors cell proliferation, cytokine secretion, and surface markers in up to 100,000 single cell co-cultures, enabling the identification of rare, functionally defined T cells, which can be recovered for clonal expansion or sequence analysis. We benchmark the platform by performing several key demonstrations. First, we show that this platform can efficiently generate monoclonal cell populations from cell lines and human T cells. Next, we demonstrate that antigen-specificity can be identified at single cell resolution using a co-culture of Jurkat cells expressing NFAT-GFP, CD8, and a T cell receptor and K562 antigen presenting cells (APC) expressing a peptide library. Thereafter, we show that immune activation in mouse and human primary samples can be monitored by time lapse analysis of Interferon gamma (IFN-{gamma}) secretion in individual microwell co-cultures using a fluorescent sandwich assay. Finally, we combine these capabilities in a proof-of-concept demonstration, which uses IFN-{gamma} secretion and the presence of CD8 surface markers as hierarchical gates to isolate and expand antigen-specific T cells from human peripheral blood, and we verify their specificity by tetramer staining. Together, these results showcase potential applications of the cloneXplorer platform in cell line development, and in screening and validating immune receptor interactions with specific antigens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/699323v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1aafc29org.highwire.dtl.DTLVardef@91272dorg.highwire.dtl.DTLVardef@1a306eeorg.highwire.dtl.DTLVardef@1bfd54_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microglia, the resident immune cells of the brain, act along a spectrum to maintain CNS homeostasis, respond to perturbations, and control neuronal activity. Disentangling the molecular mechanisms of human microglia-neuron crosstalk remains challenging due to the context-dependent, dynamic nature of their interaction. We introduce MEA-LINK, a systems-approach leveraging natural variation to screen for immune modulators of neuronal activity. This multi-modal platform integrates human induced pluripotent stem cell (hiPSC) technology with micro-electrode array (MEA) recordings and proteomic analyses of secreted immune factors, allowing for longitudinal samples and correlations across modalities. We applied MEA-LINK to explore microglia-neuron interactions during development and hyperactivity challenges. We show that human microglia accelerate neuronal network development and rescue hyperactive network phenotypes. Linking the secretome adaptations to neuronal network activity variations, we identified CCL4 as a top candidate in microglia-mediated hyperactivity control. Then, we functionally validated the context-dependent role of microglial CCL4 to neuronal CCR5 signaling in human neuronal networks. Our findings support a neuron-specific function of chemokines and their receptors in the brain and provide a new perspective for immune signaling in neuronal hyperactivity control. The MEA-LINK platform thus offers a foundation for comprehensive, systematic studies of human microglia-neuron interactions. HighlightsO_LIMEA-LINK integrates micro-electrode array recordings with proteomics of longitudinal samples to identify immune modulators of neuronal activity. C_LIO_LIHuman microglia rescue neuronal hyperactivity induced by pharmacological and genetic challenges. C_LIO_LIMicroglial CCL4 dampens neuronal activity via CCR5 signaling in a context-dependent manner. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/703799v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@18e44f7org.highwire.dtl.DTLVardef@151c450org.highwire.dtl.DTLVardef@12f7b89org.highwire.dtl.DTLVardef@57651b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding how gut microbes transform drugs, and how this influences microbiome composition and function, remains a key question to better understand the efficacy and side effects of pharmaceuticals. To accelerate the discovery of microbiome-derived drug metabolites, we developed a functional metabolomics workflow that combines the use of synthetic microbial communities (SynComs) with a time-series resolved molecular networking approach and advanced computational metabolite annotation. We demonstrate how this framework can be used to illuminate chemical transformation dynamics in a gut SynCom (Com20) with 50 clinical drugs. Our results highlight a multitude of drug metabolites, including multi-step metabolic cascades, some of which correlated to shifts in microbial taxa, suggesting functional links between microbiome composition and biochemical transformations. Our computational data analysis workflow is publicly available through the GNPS2 ecosystem at chemprop.gnps2.org, which can be used to prioritize biotransformations and other (bio)chemical reactions in various biological and abiotic systems.

Neural progenitor cell (NPC) transplantation holds immense promise for neurodegenerative and traumatic central nervous system (CNS) pathologies. However, it is crucial to define which neural circuits and pathways are targeted with transplanted NPCs under different conditions. A major roadblock lies in the limited ability to accurately trace integration of grafted cells into the host neural network. Conventional tracers suffer from drawbacks like low trans-synaptic efficiency, toxicity, and difficulty in efficiently and specifically targeting transplanted cells. To address these critical limitations, we have developed self-tracing NPCs genetically engineered to express both anterograde (WGA-mCherry) and retrograde (GFP-TTC) trans-synaptic tracers. These self-tracing NPCs maintain their intrinsic properties, differentiate into electrically active neurons, and integrate into host circuitry in vitro. Importantly, co-culture with primary rat neurons revealed successful trans-synaptic tracing of grafted human neurons, evidenced by single-positive WGA+ or TTC+ rat cells. In vivo, NPCs transplanted into a rodent spinal cord injury model retained tracer expression for 12 weeks, enabling visualization of grafted cells within the spinal cord. Co-labeling with WGA and TTC provided evidence that these NPCs forms neurons which integrated into local circuits. Our novel self-tracing NPC platform offers a powerful tool to overcome trans-synaptic tracing challenges. This approach provides the opportunity to gain critical insights into graft integration and neural circuit remodeling, paving the way for better-designed transplantation strategies and improved therapeutic outcomes in a broad spectrum of CNS disorders.

Metabolic reprogramming is a hallmark of glioblastoma, yet how distinct malignant and tumor microenvironment cell populations contribute to this metabolic heterogeneity remains poorly defined. Since direct single-cell metabolomics remains technically limited, transcriptomics-based computational inference offers a powerful alternative. Here we apply and systematically compare three complementary computational methods: (1) metabolic pathway activity scoring, (2) gene regulatory network inference focused on metabolic enzyme gene regulation, and (3) single-cell metabolic flux prediction. These methods were applied to snRNA-seq data from a set of GBM patient samples using the Human1 genome-scale metabolic model as a unified reaction and pathway annotation prior knowledge reference. Across all three methods, tumor-associated macrophages emerge as the metabolically dominant tumor microenvironment population. Tumor-associated macrophages in mesenchymal-like tumors show coordinated transcriptional control of lipid metabolism by five recurrently active transcription factors. They also exhibit consistent nucleotide biosynthesis flux and glutamate-to-glutamine conversion potentially supporting malignant cells. These findings demonstrate that multi-layered metabolic inference can resolve cell-type/state-specific dependencies in glioblastoma and highlight tumor-associated macrophage metabolism as a promising therapeutic target

Traumatic brain injury (TBI) is a major cause of mortality and long-term disability worldwide, giving rise to complex neurological complications that impact millions of individuals each year. Cellular stress and neuronal injury vary dramatically across cortical layers, vascular niches, and between the ipsilateral (injured) or contralateral (uninjured) hemispheres. There is a critical need for quantitative measures that capture the spatial distribution of injury-induced cellular changes, as well as the gene regulatory elements that drive them. Here, we developed OmicGlaze, an experimental and computational workflow for systematically profiling the spatial transcriptome and epigenome of mouse brains following mild traumatic brain injury. We established a spatial scoring system, and identified region-specific biological processes post injury, including changes in neuronal activities, cellular stress, immune response, and gliosis. Spatial assay for transposase-accessible chromatin with sequencing (Spatial ATAC-seq) generated the first epigenetic map of traumatic brain injury near single-cell resolution. Notably, we identified the Activator Protein-1 family transcription factor Atf3 as a key gene regulator of injury-induced cellular stress. Together, these spatial multi-omics analyses revealed gene regulatory network in TBI and provided a broadly applicable framework for dissecting cellular and molecular mechanisms underlying complex neurological disorders.

Genetic engineering of primary hematopoietic cells is essential for mechanistic immunology studies, however the development of cell-based therapies yet remains constrained by two major factors: the fragility of many primary lineages and challenges of viral delivery platforms that are costly, time-intensive, and biologically confounding. Here, we optimized scalable, non-viral CRISPR-Cas9 electroporation workflows using the ExPERT platform across three primary mouse hematopoietic cell types: OT-I CD8 T cells, bone marrow-derived macrophages (BMDMs), and hematopoietic stem cells (HSCs). In activated OT-I CD8 T cells, two electroporation programs supported high-efficiency mRNA and RNP delivery with minimal impact on cell viability or proliferative capacity, with subtle activation-state-dependent sensitivity at higher energy settings. Extending optimization to myeloid and stem cell lineages, we found that BMDMs maintained high viability following electroporation, and a high-performing electroporation program supported robust RNP delivery and efficient target gene knockout while preserving macrophage differentiation. In HSCs, the same program enabled consistent RNP delivery, sustained viability, and reproducible gene knockout. Together, these findings establish ExPERT electroporation as a robust, reproducible, and modular platform for non-viral genome editing across primary mouse hematopoietic lineages, lowering barriers to rapid genetic perturbation for both discovery and translational applications.