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This study investigates the impact of sodium channel protein type 1 subunit alpha (SCN1A) gene knockout (SCN1A KO) on brain development and function using cerebral organoids coupled with a multiomics approach. From comprehensive omics analyses,we found that SCN1A KO organoids exhibit decreased growth,dysregulated neurotransmitter levels,and altered lipidomic,proteomic,and transcriptomic profiles compared to controls under matrix-free differentiation conditions. Neurochemical analysis reveals reduced levels of key neurotransmitters,and lipidomic analysis highlights changes in ether phospholipids and sphingomyelin. Furthermore,quantitative profiling of the SCN1A KO organoid proteome shows perturbations in cholesterol metabolism and sodium ion transportation,potentially affecting synaptic transmission. These findings suggest dysregulation of cholesterol metabolism and sodium ion transport,with implications for synaptic transmission. Overall,these insights shed light on the molecular mechanisms underlying SCN1A-associated disorders,such as Dravet syndrome,and offer potential avenues for therapeutic intervention. View Publication

This study presents a comprehensive transcriptomic analysis of feeder-free extended pluripotent stem cells (ffEPSCs) and their parental human embryonic stem cells (ESCs),providing new insights into understanding human early development and cellular heterogeneity of pluripotency. Leveraging Smart-seq2-based single-cell RNA sequencing (scRNA-seq),we have compared gene expression profiles between ESCs and ffEPSCs and uncovered distinct subpopulations within both groups. Through pseudotime analysis,we have mapped the transition process from ESCs to ffEPSCs,revealing critical molecular pathways involved in the shift from a primed pluripotency to an extended pluripotent state. Additionally,we have employed repeat sequence analysis based on the latest T2T database and identified the stage-specific repeat elements contributing to regulating pluripotency and developmental transitions. This dataset deepens our understanding on early pluripotency and highlights the role of repeat sequences in early embryonic development. Our findings thus offer valuable resources for researchers in stem cell biology,pluripotency,early embryonic development,and potential cell therapy and regenerative medical applications. View Publication

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs),yet their role in MNs remains unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for other hematopoietic lineages. Ddx41 knockout in early erythropoiesis is embryonically lethal,while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4),which co-distribute with DDX41 on the erythroid genome. DDX41 directly binds to and resolves G4,which is significantly compromised in MN-associated DDX41 mutants. G4 accumulation induces erythroid genome instability,ribosomal defects,and p53 upregulation. However,p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel,genome instability also activates the cGas-Sting pathway,impairing survival,as cGas deficiency rescues the lethality of hematopoietic-specific Ddx41 knockout mice. This is supported by data from a DDX41-mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 resolvase,essential for erythroid genome stability and suppressing the cGAS-STING pathway. Germline DDX41 mutations are linked to myeloid neoplasms,but their roles in the disease is unclear. Here,the authors show that DDX41 resolves G-quadruplex structures to maintain erythroid genome stability and prevent cGAS-mediated cell death. These functions are lost in disease-associated variants. View Publication

AbstractNeurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially reduce accumulation of neurological deficits and conversion to progressive disease forms. Diet and the associated gut microbiome are currently being discussed as crucial environmental risk factors that determine disease onset and subsequent progression. In people with multiple sclerosis,supplementation of the short-chain fatty acid propionic acid,as a microbial metabolite derived from the fermentation of a high-fiber diet,has previously been shown to regulate inflammation accompanied by neuroprotective properties. We set out to determine whether the neuroprotective impact of propionic acid is a direct mode of action of short-chain fatty acids on CNS neurons. We analysed neurite recovery in the presence of the short-chain fatty acid propionic acid and butyric acid in a reverse-translational disease-in-a-dish model of human-induced primary neurons differentiated from people with multiple sclerosis-derived induced pluripotent stem cells. We found that recovery of damaged neurites is induced by propionic acid and butyric acid. We could also show that administration of butyric acid is able to enhance propionic acid-associated neurite recovery. Whole-cell proteome analysis of induced primary neurons following recovery in the presence of propionic acid revealed abundant changes of protein groups that are associated with the chromatin assembly,translational,and metabolic processes. We further present evidence that these alterations in the chromatin assembly were associated with inhibition of histone deacetylase class I/II following both propionic acid and butyric acid treatment,mediated by free fatty acid receptor signalling. While neurite recovery in the presence of propionic acid is promoted by activation of the anti-oxidative response,administration of butyric acid increases neuronal ATP synthesis in people with multiple sclerosis-specific induced primary neurons. In human multiple sclerosis-specific neurons,differentiated via induced pluripotent stem cells,Gisevius et al. display neuroregeneration mediated by the short-chain fatty acids propionic and butyric acid. Intracellularly,free fatty acid receptor signalling leads to inhibition of histone deacetylase activity,thereby altering the oxidative stress response and cellular protein biosynthesis. Graphical Abstract Graphical Abstract View Publication

ABSTRACTLoss of Cdx2 in vivo leads to stunted development of the allantois,an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here,we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT),heterozygous (CDX2-Het),and homozygous null CDX2 (CDX2-KO) genotypes,differentiating these cells in a 2D gastruloid model,and subjecting these cells to single-nucleus RNA and ATAC sequencing,we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile,suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo,we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together,these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo. Summary: Using 2D human gastruloids,CDX2 is shown to dose-dependently influence genes related to tissue permeability,cell-cell adhesions,and cytoskeletal architecture during extraembryonic mesoderm development. View Publication

Astroglia are integral to brain development and the emergence of neurodevelopmental disorders. However,studying the pathophysiology of human astroglia using brain organoid models has been hindered by inefficient astrogliogenesis. In this study,we introduce a robust method for generating astroglia-enriched organoids through BMP4 treatment during the neural differentiation phase of organoid development. Our RNA sequencing analysis reveals that astroglia developed within these organoids exhibit advanced developmental characteristics and enhanced synaptic functions compared to those grown under traditional two-dimensional conditions,particularly highlighted by increased neurexin (NRXN)-neuroligin (NLGN) signaling. Cell adhesion molecules,such as NRXN and NLGN,are essential in regulating interactions between astroglia and neurons. We further discovered that brain organoids derived from human embryonic stem cells (hESCs) harboring the autism-associated NLGN3 R451C mutation exhibit increased astrogliogenesis. Notably,the NLGN3 R451C astroglia demonstrate enhanced branching,indicating a more intricate morphology. Interestingly,our RNA sequencing data suggest that these mutant astroglia significantly upregulate pathways that support neural functions when compared to isogenic wild-type astroglia. Our findings establish a novel astroglia-enriched organoid model,offering a valuable platform for probing the roles of human astroglia in brain development and related disorders.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-024-00219-5. View Publication

Understanding how embryonic progenitors decode extrinsic signals and transform into lineage-specific regulatory networks to drive cell fate specification is a fundamental,yet challenging question. Here,we develop a new model of surface epithelium (SE) differentiation induced by human embryonic stem cells (hESCs) using retinoic acid (RA),and identify BMP4 as an essential downstream signal in this process. We show that the retinoid X receptors,RXRA and RXRB,orchestrate SE commitment by shaping lineage-specific epigenetic and transcriptomic landscapes. Moreover,we find that TCF7,as a RA effector,regulates the transition from pluripotency to SE initiation by directly silencing pluripotency genes and activating SE genes. MSX2,a downstream activator of TCF7,primes the SE chromatin accessibility landscape and activates SE genes. Our work reveals the regulatory hierarchy between key morphogens RA and BMP4 in SE development,and demonstrates how the TCF7-MSX2 axis governs SE fate,providing novel insights into RA-mediated regulatory principles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05525-4. View Publication

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade,an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders,the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing,we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay,intellectual disability,microcephaly,and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary,we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder. View Publication

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells,followed by the migration,differentiation,and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens,such as FGF8. Disruption of this developmental balance can lead to brain malformations,which underlie a range of complex neurodevelopmental disorders,including epilepsy,autism,and intellectual disabilities. Studying the early stages of human brain development,whether under normal or pathological conditions,remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently,human brain organoids have emerged as a powerful in vitro alternative,allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures,where neural progenitors and neurons are grown on flat surfaces,3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However,3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore,few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning.• To address these limitations,we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs),our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation,NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors,where they self-organize into neural rosettes and neuroepithelial structures,surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids,leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity,enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors,followed by dissociation and reaggregation into 3D brain organoids.• For optimal growth and maturation,telencephalic organoids are cultured in spinning mini-bioreactors [2] or on orbital shakers.• The protocol enables the generation of telencephalic neural progenitors in 10 days and produces 3D telencephalic organoids containing neocortical neurons within one month of culture.• Addition of morphogens in the culture medium (example: FGF8) enhances cellular heterogeneity,promoting the emergence of distinct brain domains within a single organoid. View Publication

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of hepatic disorders,ranging from simple steatosis to steatohepatitis,with the most severe outcomes including cirrhosis,liver failure,and hepatocellular carcinoma. Notably,MASLD prevalence is lower in premenopausal women than in men,suggesting a potential protective role of estrogens in mitigating disease onset and progression. In this study,we utilized preclinical in vitro models—immortalized cell lines and hepatocyte-like cells derived from human embryonic stem cells—exposed to clinically relevant steatotic-inducing agents. These exposures led to lipid droplet (LD) accumulation,increased reactive oxygen species (ROS) levels,and mitochondrial dysfunction,along with decreased expression of markers associated with hepatocyte functionality and differentiation. Estrogen treatment in steatotic-induced liver cells resulted in reduced ROS levels and LD content while preserving mitochondrial integrity,mediated by the upregulation of mitochondrial thioredoxin 2 (TRX2),an antioxidant system regulated by the estrogen receptor. Furthermore,disruption of TRX2,either pharmacologically using auranofin or through genetic interference,was sufficient to counteract the protective effects of estrogens,highlighting a potential mechanism through which estrogens may prevent or slow MASLD progression. View Publication

Background: Pulmonary fibrosis is a major disease that leads to the progressive loss of lung function. The disease manifests early,resulting in type 2 respiratory failure. This is likely due to the bronchocentric fibrosis around the major airways,which causes airflow limitation. It affects approximately three million patients worldwide and has a poor prognosis. Skin fibroblasts isolated from patients offer valuable insights into understanding the disease mechanisms,identifying the genetic causes,and developing personalized therapies. However,the use of skin fibroblasts to study a disease that exclusively impacts the lungs is often questioned,particularly since lung fibrosis primarily affects the alveolar epithelium. Method: We report the reprogramming of skin fibroblasts from patients with an atypical early-onset form of lung fibrosis into induced pluripotent stem cells (iPSCs) and subsequently into alveolar epithelial cells. This was achieved using a Sendai virus approach. Results: We show that the reprogrammed cells carry mutations in the calcium-binding protein genes S100A3 and S100A13,leading to diminished protein expression,thus mimicking the patients’ cells. Additionally,we demonstrate that the generated patient iPSCs exhibit aberrant calcium and mitochondrial functions. Conclusions: Due to the lack of a suitable animal model that accurately resembles the human disease,generating patient lung cells from these iPSCs can provide a valuable “disease in a dish” model for studying the atypical form of inherited lung fibrosis. This condition is associated with mutations in the calcium-binding protein genes S100A3 (NM_002960) and S100A13 (NM_001024210),aiding in the understanding of its pathogenesis. View Publication

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types,such as cardiac fibroblasts,into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore,the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs,incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis. View Publication