技术资料

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart,lungs,kidney,and brain are all hallmarks of the disease,the most severe symptoms of TSC are often neurological,including seizures,autism,psychiatric disorders,and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented,it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes,we observed aberrant early neurodevelopment in vitro,including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions,including loci associated with key genes in neurodevelopment. Collectively,these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated,with implications for whether and how prenatal treatment should be pursued. View Publication

Human pancreatic islets regulate organ development and metabolic homeostasis,with dysfunction leading to diabetes. Human pluripotent stem cells (hPSCs) provide a potential alternative source to cadaveric human pancreatic islets for replacement therapy in diabetes. However,human islet-like organoids (HILOs) generated from hPSCs in vitro often exhibit heterogeneous immature phenotypes such as aberrant gene expression and inadequate insulin secretion in response to glucose. Here we show that FXYD Domain Containing Ion Transport Regulator 2 (FXYD2) marks and regulates functional maturation and heterogeneity of generated HILOs,by controlling the ? cell transcriptome necessary for glucose-stimulated insulin secretion (GSIS). Despite its presence in mature ? cells,FXYD2 is diminished in hPSC-derived ?-like cells. Mechanistically,we find that FXYD2 physically interacts with SRC proto-oncogene,non-receptor tyrosine kinase (SRC) protein to regulate FXYD2-SRC-TEAD1 signaling to modulate ? cell transcriptome. We demonstrate that FXYD2High HILOs significantly outperform FXYD2Low counterparts to improve hyperglycemia in STZ-induced diabetic immune deficient mice. These results suggest that FXYD2 marks and regulates human ? cell maturation via channel-sensing signal transduction and that it can be used as a selection marker for functional heterogeneity of stem cell derived human islet organoids. Tacto et al. uncover a key marker that enables the selection of functional,transplantable human islets derived from stem cells,potentially paving the way for more precise and effective diabetes cell therapy. View Publication

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity,leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity,leaving a gap in the comprehensive understanding of clonal heterogeneity. Here,we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic,epigenomic,endogenous retroelement,and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs—productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion,accompanied by elevated levels of phosphorylated ERK1/2. Conversely,PC exhibited enrichment in embryonic organ development and transcription factor activation,accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably,TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones,thereby mitigating adverse outcomes in clinical applications.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05493-9. View Publication

SummaryMolecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation,presence of a putative ciliary margin zone,emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data,we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development,and to show that chromatin accessibility is highly correlated to the developing human retina,but with some differences in the temporal emergence and abundance of some of the retinal neurons. Graphical abstract Highlights•Single cell analyses reveal putative ciliary margin (pCM) presence in retinal organoids•PCM harbors early RPCs which differentiate to late RPCs and retinal neurons•Single cell ATAC-seq data reveal novel TF binding motifs in RPCs and retinal neurons•RO development largely recapitulates retinogenesis Genetics; Molecular biology; Neuroscience; Cell biology; Omics View Publication

Using live-cell microscopy,we find that loss of VPS13C in human neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal tethers,leading to impaired lysosomal motility and defective lysosomal function as well as a decreased phospho-Rab10-mediated lysosomal stress response. Loss-of-function mutations in VPS13C are linked to early-onset Parkinson’s disease (PD). While VPS13C has been previously studied in non-neuronal cells,the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy,we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts,leading to impaired lysosomal motility and cellular distribution,as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD. View Publication

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