GABAergic neuronal lineage development determines clinically actionable targets in diffuse hemispheric glioma, H3G34-mutant.

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target.

Joint single-cell genetic and transcriptomic analysis reveal pre-malignant SCP-like subclones in human neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. It is a complex disease that can result in diverse clinical outcomes. In some children, tumors regress spontaneously. Others respond well to existing treatments. But for the high-risk group, which constitutes approximately 40% of all patients, the prognosis remains dire despite collaborative efforts in basic and clinical research. While its exact cellular origin is still under debate, NB is assumed to arise from the neural crest cell lineage including multipotent Schwann cell precursors (SCPs), which differentiate into sympatho-adrenal cell states eventually producing chromaffin cells and sympathoblasts. METHODS: To investigate clonal development of neuroblastoma cell states, we performed haplotype-specific analysis of human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Samples were also assessed using immunofluorescence stainings and fluorescence in-situ hybridization (FISH). RESULTS: Beyond adrenergic tumor cells, we identify subpopulations of aneuploid SCP-like cells, characterized by clonal expansion, whole-chromosome 17 gains, as well as expression programs of proliferation, apoptosis, and a non-immunomodulatory phenotype. CONCLUSION: Aneuploid pre-malignant SCP-like cells represent a novel feature of NB. Genetic evidence and tumor phylogeny suggest that these clones and malignant adrenergic populations originate from aneuploidy-prone cells of migrating neural crest or SCP origin, before lineage commitment to sympatho-adrenal cell states. Our findings expand the phenotypic spectrum of NB cell states. Considering the multipotency of SCPs in development, we suggest that the transformation of fetal SCPs may represent one possible mechanism of tumor initiation in NB with chromosome 17 aberrations as a characteristic element.

Chromatin accessibility during human first-trimester neurodevelopment.

The human brain develops through a tightly organized cascade of patterning events, induced by transcription factor expression and changes in chromatin accessibility. Although gene expression across the developing brain has been described at single-cell resolution1, similar atlases of chromatin accessibility have been primarily focused on the forebrain2-4. Here we describe chromatin accessibility and paired gene expression across the entire developing human brain during the first trimester (6-13 weeks after conception). We defined 135 clusters and used multiomic measurements to link candidate cis-regulatory elements to gene expression. The number of accessible regions increased both with age and along neuronal differentiation. Using a convolutional neural network, we identified putative functional transcription factor-binding sites in enhancers characterizing neuronal subtypes. We applied this model to cis-regulatory elements linked to ESRRB to elucidate its activation mechanism in the Purkinje cell lineage. Finally, by linking disease-associated single nucleotide polymorphisms to cis-regulatory elements, we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder-related mutations. Our findings provide a more detailed view of key gene regulatory mechanisms underlying the emergence of brain cell types during the first trimester and a comprehensive reference for future studies related to human neurodevelopment.

Comprehensive cell atlas of the first-trimester developing human brain.

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin.

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

Stem Cell Therapies for Central Nervous System Trauma: The 4 Ws-What, When, Where, and Why.

Traumatic injury of the central nervous system (CNS) is a worldwide health problem affecting millions of people. Trauma of the CNS, that is, traumatic brain injury (TBI) and spinal cord injury (SCI), lead to massive and progressive cell loss and axonal degeneration, usually with very limited regeneration. At present, there are no treatments to protect injured CNS tissue or to replace the lost tissue. Stem cells are a cell type that by definition can self-renew and give rise to multiple cell lineages. In recent years, therapies using stem and progenitor cells have shown promising effects in experimental CNS trauma, particularly in the acute-subacute stage, but also in chronic injuries. However, the therapeutic mechanisms by which transplanted cells achieve the structural and/or functional improvements are often not clear. Stem cell therapies for CNS trauma can be categorized into 2 main concepts, transplantation of exogenous neural stem cells and neural progenitor cells and recruitment of endogenous stem and progenitor cells. In this review, focusing on the advances during the last decade, we will discuss the major cell therapies, the pros and cons of these 2 concepts for TBI and SCI, and the treatment strategies we believe will be successful.

Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

Multiple therapeutic effects of human neural stem cells derived from induced pluripotent stem cells in a rat model of post-traumatic syringomyelia.

BACKGROUND: Post-traumatic syringomyelia (PTS) affects patients with chronic spinal cord injury (SCI) and is characterized by progressive deterioration of neurological symptoms. To improve surgical treatment, we studied the therapeutic effects of neuroepithelial-like stem cells (NESCs) derived from induced pluripotent stem cells (iPSCs) in a rat model of PTS. To facilitate clinical translation, we studied NESCs derived from Good Manufacturing Practice (GMP)-compliant iPSCs. METHODS: Human GMP-compliant iPSCs were used to derive NESCs. Cryo-preserved NESCs were used off-the-shelf for intraspinal implantation to PTS rats 1 or 10 weeks post-injury, and rats were sacrificed 10 weeks later. In vivo cyst volumes were measured with micro-MRI. Phenotypes of differentiated NESCs and host responses were analyzed by immunohistochemistry. FINDINGS: Off-the-shelf NESCs transplanted to PTS rats 10 weeks post-injury reduced cyst volume. The grafted NESCs differentiated mainly into glial cells. Importantly, NESCs also stimulated tissue repair. They reduced the density of glial scars and neurite-inhibiting chondroitin sulfate proteoglycan 4 (CSPG4), stimulated host oligodendrocyte precursor cells to migrate and proliferate, reduced active microglia/macrophages, and promoted axonal regrowth after subacute as well as chronic transplantation. INTERPRETATION: Significant neural repair promoted by NESCs demonstrated that human NESCs could be used as a complement to standard surgery in PTS. We envisage that future PTS patients transplanted with NESCs will benefit both from eliminating the symptoms of PTS, as well as a long-term improvement of the neurological symptoms of SCI. FUNDING: This work was supported by Vinnova (2016-04134), Karolinska Institutet StratRegen, and the Chinese Scholarship Council.

In Vitro Study of Human Immune Responses to Hyaluronic Acid Hydrogels, Recombinant Spidroins and Human Neural Progenitor Cells of Relevance to Spinal Cord Injury Repair.

Scaffolds of recombinant spider silk protein (spidroin) and hyaluronic acid (HA) hydrogel hold promise in combination with cell therapy for spinal cord injury. However, little is known concerning the human immune response to these biomaterials and grafted human neural stem/progenitor cells (hNPCs). Here, we analyzed short- and long-term in vitro activation of immune cells in human peripheral blood mononuclear cells (hPBMCs) cultured with/without recombinant spidroins, HA hydrogels, and/or allogeneic hNPCs to assess potential host-donor interactions. Viability, proliferation and phenotype of hPBMCs were analyzed using NucleoCounter and flow cytometry. hPBMC viability was confirmed after exposure to the different biomaterials. Short-term (15 h) co-cultures of hPBMCs with spidroins, but not with HA hydrogel, resulted in a significant increase in the proportion of activated CD69+ CD4+ T cells, CD8+ T cells, B cells and NK cells, which likely was caused by residual endotoxins from the Escherichia coli expression system. The observed spidroin-induced hPBMC activation was not altered by hNPCs. It is resource-effective to evaluate human compatibility of novel biomaterials early in development of the production process to, when necessary, make alterations to minimize rejection risk. Here, we present a method to evaluate biomaterials and hPBMC compatibility in conjunction with allogeneic human cells.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Transplantation of Human Neural Precursor Cells Reverses Syrinx Growth in a Rat Model of Post-Traumatic Syringomyelia.

Posttraumatic syringomyelia (PTS) is a serious condition of progressive expansion of spinal cord cysts, affecting patients with spinal cord injury years after injury. To evaluate neural cell therapy to prevent cyst expansion and potentially replace lost neurons, we developed a rat model of PTS. We combined contusive trauma with subarachnoid injections of blood, causing tethering of the spinal cord to the surrounding vertebrae, resulting in chronically expanding cysts. The cysts were usually located rostral to the injury, extracanalicular, lined by astrocytes. T2*-weighted magnetic resonance imaging (MRI) showed hyperintense fluid-filled cysts but also hypointense signals from debris and iron-laden macrophages/microglia. Two types of human neural stem/progenitor cells-fetal neural precursor cells (hNPCs) and neuroepithelial-like stem cells (hNESCs) derived from induced pluripotent stem cells-were transplanted to PTS cysts. Cells transplanted into cysts 10 weeks after injury survived at least 10 weeks, migrated into the surrounding parenchyma, but did not differentiate during this period. The cysts were partially obliterated by the cells, and cyst walls often merged with thin layers of cells in between. Cyst volume measurements with MRI showed that the volumes continued to expand in sham-transplanted rats by 102%, while the cyst expansion was effectively prevented by hNPCs and hNESCs transplantation, reducing the cyst volumes by 18.8% and 46.8%, respectively. The volume reductions far exceeded the volume of the added human cells. Thus, in an animal model closely mimicking the clinical situation, we provide proof-of-principle that transplantation of human neural stem/progenitor cells can be used as treatment for PTS.

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution.

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

Non-specific Detection of a Major Western Blotting Band in Human Brain Homogenates by a Multitude of Amyloid Precursor Protein Antibodies.

The use of human post-mortem brain material is of great value when investigating which pathological mechanisms occur in human brain, and to avoid translational problems which have for example been evident when translating animal research into Alzheimer disease (AD) clinical trials. The amyloid ß (Aß)-peptide, its amyloid precursor protein (APP) and the intermediate APP-c-terminal fragments (APP-CTFs) are all important players in AD pathogenesis. In order to elucidate which APP CTF that are the most common in brain tissue of different species and developmental stages, and whether there are any differences in these fragments between AD and control brain, we investigated the occurrence of these fragments using different APP c-terminal antibodies. We noticed that whereas the conventional APP-CTFα and CTFß fragments were most prominent in rat and mouse brain tissue, the major western blotting band detected in human, macaque and guinea pig was of approximately 20 kDa in size, possibly corresponding to the newly discovered APP-CTFη. However, this band was also intensely stained with a total protein stain, as well as by several other antibodies. The staining intensity of the 20 kDa band by the APP antibodies varied considerably between samples and correlated with the staining intensity of this band by the total protein stain. This could potentially be due to non-specific binding of the antibodies to another protein of this size. In-gel digestion and mass spectrometry confirmed that small amounts of APP were present in this band, but many other proteins were identified as well. The major hit of the mass spectrometry analysis was myelin basic protein (MBP) and a myelin removal protocol removed proportionally more of the 20 kDa APP band than the full-length APP and APP-CTFα/ß bands. However, the signal could not be immunodepleted with an MBP antibody. In summary, we report on a potentially non-specific western blotting band of approximately 20 kDa and call for precaution when analyzing proteins of this size in human brain tissue.

Identification of amino acid residues of nerve growth factor important for neurite outgrowth in human dorsal root ganglion neurons.

Nerve growth factor (NGF) is an essential neurotrophic factor for the development and maintenance of the central and the peripheral nervous system. NGF deficiency in the basal forebrain precedes degeneration of basal forebrain cholinergic neurons in Alzheimer's disease, contributing to memory decline. NGF mediates neurotrophic support via its high-affinity receptor, the tropomyosin-related kinase A (TrkA) receptor, and mediates mitogenic and differentiation signals via the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). However, the molecular mechanisms underlying the different NGF/TrkA/ERK signalling pathways are far from clear. In this study, we have investigated the role of human NGF and three NGF mutants, R100E, W99A and K95A/Q96A, their ability to activate TrkA or ERK1/2, and their ability to induce proliferation or differentiation in human foetal dorsal root ganglion (DRG) neurons or in PC12 cells. We show that the R100E mutant was significantly more potent than NGF itself to induce proliferation and differentiation, and significantly more potent in activation of ERK1/2 in DRG neurons. The W99A and K95A/Q96A mutants, on the other hand, were less effective than the wild-type protein. An unexpected finding was the high efficacy of the K95A/Q96A mutant to activate TrkA and to induce differentiation of DRG neurons at elevated concentrations. These data demonstrate an NGF mutant with improved neurotrophic properties in primary human neuronal cells. The R100E mutant represents an interesting candidate for further drug development in Alzheimer's disease and other neurodegenerative disorders.

Termination of cell-type specification gene programs by the miR-183 cluster determines the population sizes of low-threshold mechanosensitive neurons.

Touch and mechanical sensations require the development of several different kinds of sensory neurons dedicated to respond to certain types of mechanical stimuli. The transcription factor Shox2 (short stature homeobox 2) is involved in the generation of TRKB+ low-threshold mechanoreceptors (LTMRs), but mechanisms terminating this program and allowing alternative fates are unknown. Here, we show that the conditional loss of the miR-183-96-182 cluster in mouse leads to a failure of extinction of Shox2 during development and an increase in the proportion of Aδ LTMRs (TRKB+/NECAB2+) neurons at the expense of Aß slowly adapting (SA)-LTMRs (TRKC+/Runx3-) neurons. Conversely, overexpression of miR-183 cluster that represses Shox2 expression, or loss of Shox2, both increase the Aß SA-LTMRs population at the expense of Aδ LTMRs. Our results suggest that the miR-183 cluster determines the timing of Shox2 expression by direct targeting during development, and through this determines the population sizes of Aδ LTMRs and Aß SA-LTMRs.

RNA velocity of single cells.

RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFß expression.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT-PCR analysis, we observed increased Transforming growth factor-ß (TGFß) gene expression in CADASIL VSMCs. Adding TGFß-neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFß-neutralizing antibody in ECs co-cultured with VSMCs. ECs co-cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co-cultured with control VSMCs, and neutralization of TGFß normalized the proliferation rate of ECs co-cultured with CADASIL VSMCs. We suggest that increased TGFß expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.