技术资料

Rett syndrome arises from loss-of-function mutations in the X-linked chromatin regulator MECP2,yet the earliest molecular derailments in development are poorly defined. Using isogenic human embryonic stem cell (hESC) models carrying three patient-derived MECP2 mutations,we followed the transcriptome from pluripotency through neuroectoderm,neural stem/progenitor stages. Developmental stage dominated transcriptional variance,but mutants shared a secondary program enriched for synaptic-membrane and extracellular matrix genes. Single-cell/bulk profiling at the embryonic stem cell (ESC) stage revealed partial naïve-like drift,marked by the up-regulation of the naïve-enriched factor ZFP42/REX1 and related markers in MECP2-mutant lines. Among convergently dysregulated genes,the cortical determinant EMX1 showed an abnormal developmental trajectory,early repression followed by overshoot,and was consistently altered across independent Rett PSC models. Single-nucleus RNA-seq of cerebral organoids uncovered allele-specific yet convergent disturbances in cortical lineage allocation. These data chart a continuous developmental trajectory for MECP2-mutant cells and nominate naïve-like drift and mis-timed EMX1 expression as tractable entry points for dissecting Rett pathogenesis. Graphical abstract Highlights•MECP2 mutations induce an early naïve-like transcriptional drift in hESCs•EMX1 shows a conserved abnormal developmental trajectory across Rett models•Shared transcriptional programs emerge during neural induction in MECP2 mutants•Rett cerebral organoids display mutation-specific shifts in lineage allocation In this article,Flamier and colleagues show that MECP2 mutations perturb human neurodevelopment from the pluripotent stage onward. Using isogenic hESC and organoid models,they identify an early naïve-like transcriptional drift,abnormal EMX1 timing,and convergent defects in cortical lineage allocation,revealing continuous developmental vulnerability in Rett syndrome. View Publication

The interaction between human respiratory syncytial virus (HRSV) and the innate immune system has been demonstrated both in vitro and in vivo. Disruption of interferon (IFN) signaling pathways increases susceptibility and permissiveness to HRSV infection,whereas pretreatment of cells with IFN confers (partial) resistance. This suggests that HRSV disease severity is likely influenced by a pre-existing antiviral state of the respiratory epithelium,driven by baseline or primed expression of type I and type III IFNs. Here,we investigated whether prior exposure to respiratory bacteria or viruses alters in vitro susceptibility and permissiveness to HRSV infection by shaping an antiviral state using both immortalized cell lines and airway organoid models. In A549 cells,pre-exposure to S. aureus had the most significant impact by reducing HRSV infection and inducing robust interferon responses. However,this effect was not reproduced in airway organoids. Conversely,sequential virus infection experiments in airway organoids revealed that prior infection with human parainfluenza virus type 3 (HPIV‑3) reduced the spread of subsequent HRSV infection. In addition to interferon signaling this proved to be associated with epithelial damage mediated by HPIV-3 infection. Collectively,these findings show that HRSV susceptibility and permissiveness are influenced by the up- or downregulation of specific anti- and pro-viral factors induced by prior bacterial or viral exposure,together with the maintenance or disruption of epithelial integrity. Understanding these interactions could be crucial when identifying specific risk groups for severe HRSV-associated disease and the development of targeted HRSV interventions. View Publication

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However,methodologies for isolating functional synaptic structures from these models remain limited. Here,we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air‐liquid interface cerebral organoids (ALI‐COs) at distinct developmental stages (Day 90 and 150). Notably,the method avoids density gradients,requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g.,GAP43) and classical synaptosomal proteins (e.g.,PCLO,BSN,SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane‐enclosed GCPs with fibrous content and mitochondria in Day 90 isolates,and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl‐induced depolarization,which triggered phosphorylation changes in growth cone proteins (GAP43,MARCKS,MARCKSL1),cytoskeletal regulators (DCLK1,SHTN1,MARK4,MAP1B) and protein kinases (CAMK2G,PRKCE) in Day 90 GCPs,as well as classical synaptic vesicle cycle proteins (SYN1,DNM1,RPH3A) at Day 150. Overall,this study establishes a centrifugation‐based protocol for isolating growth cones and immature synapses from human organoids,capturing key stages of synaptic development and enabling scalable,patient‐compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders. Synapses are implicated in several neurological disorders and psychiatric diseases. The emergence and wide use of neural organoids provide a new opportunity to study human synapses in healthy and disease settings. Therefore,we developed a simple method for the enrichment of synaptosomes and growth cone particles from forebrain organoids. The method is based on differential centrifugation,works with small tissue amounts,and is highly reproducible. We validated the functionality of the isolated structures using KCl stimulation and phosphoproteomics. The method enables detailed mapping of protein composition and function during growth cone pathfinding,synaptogenesis,and establishment of neural circuits in organoids. View Publication

Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases,but relevant human models are missing. Here,we present a robust 3D in vitro method with the use of human induced pluripotent stem cell–derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing,high-resolution immunofluorescence,and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells,pericytes,and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O2) for 1 week recapitulated key pathological features,including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases. View Publication

Progressive supranuclear palsy-Richardson’s syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however,the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here,we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation,neurofilament accumulation,and structural alterations of the axonal compartment,consistent with an early axonopathic phenotype. Notably,mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels,indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion,our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation,impaired structural homeostasis,and altered signaling pathways. Rather than acting as a primary driver,mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS. View Publication