参考文献

Stem cell therapy has been widely studied as a promising treatment for spinal cord injury (SCI). However,a lack of functional scaffolds for stem cell therapy to address SCI leads to low therapeutic efficacy due to poor survival of transplanted cells. To address these challenges,this study aims to enhance regenerative potential of spinal cord organoids (SCOs) by employing extracellular matrix (ECM) recapitulating spinal cord-specific microenvironment. Decellularized spinal cord-derived ECM (ScEM) supports 3D culture for development,maturation,and functionality of human induced pluripotent stem cell-derived SCOs,comparable to standard organoid culture matrix such as Matrigel. Transplantation of SCOs using ScEM hydrogel promotes axonal regeneration with neovascularization in lesions,likely because of enhanced engraftment and integration of transplanted SCOs into defective tissues facilitated by ScEM. Accordingly,this approach induces early locomotor recovery of animals with SCI. These findings suggest that functional ECM scaffold capable of providing microenvironmental complexity of spinal cord can potentiate organoid-based therapeutics for SCI treatment. Graphical abstract Highlights•A decellularized spinal cord-derived ECM (ScEM) exhibits protein profiles resembling native spinal cord tissue.•Functional proteins related to neurodevelopment and regeneration are highly enriched in ScEM.•ScEM hydrogel supports development and maturation of spinal cord organoids (SCOs) comparable to Matrigel.•ScEM hydrogel enhances SCO-mediated axonal regrowth,immune modulation,and early recovery after spinal cord injury. View Publication

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system,but it involutes with age,resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro,but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However,these approaches require access to primary human tissues or murine bone marrow stromal cells,are allogeneic,and do not support negative selection. Recently,we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here,we combined hPSC-derived TEPs,hematopoietic progenitor cells (HPCs),and mesenchymal cells,differentiated from the same hPSC line,and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic,patient-specific thymic organoids in vitro. 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

Background: Age-related macular degeneration (AMD) is a leading cause of vision loss. Reticular pseudodrusen (RPD),deposits on the apical side of the retinal pigment epithelium (RPE),signify a distinctive and critical AMD phenotype. Yet,their molecular basis and relationship to the conventional drusen seen in AMD remain unclear. Methods: We generated induced pluripotent stem cell-derived RPE cells from a clinically phenotyped cohort comprising only individuals with conventional drusen (AMD/RPD-) or with drusen coexisting with RPD (AMD/RPD +). To identify differences between the two cohorts,we performed single-cell transcriptomic,proteomic,quantitative trait locus (QTL) and transcriptome-wide association (TWAS) analyses,together with functional assays. Results: AMD/RPD + RPE cells exhibited enrichment of extracellular matrix (ECM) and hypoxia-responsive pathways,and a relative underrepresentation of mitochondrial and oxidative phosphorylation processes,when compared with AMD/RPD- cells. Genetic analyses supported shared modulation of mitochondrial pathways across AMD,with additional regulatory signals associated with RPD risk. Functionally,all RPE cohorts formed drusen-like deposits in vitro. AMD/RPD- lines generated more basal deposits,whereas AMD/RPD + cells exhibited increased susceptibility to monolayer disruption. Conclusions: These findings indicate that AMD with and without RPD represent mechanistically distinct entities and provide novel insight into the molecular mechanisms underlying disease heterogeneity in AMD. View Publication

Genome‐wide association studies (GWAS) have identified APOE2 allele as linked to exceptional longevity,with carriers exhibiting a reduced risk of Alzheimer's disease (AD). Apolipoprotein E (APOE),a glycoprotein involved in lipid transport,has three major alleles. However,alterations in lipid metabolism alone do not fully explain APOE2's protective effects. In contrast,APOE4 is the strongest genetic risk factor for AD. To investigate how APOE2 promotes neuronal longevity and confers neuroprotection,we generated human isogenic APOE iPSC‐derived models of both inhibitory GABAergic and excitatory neurons. In GABAergic neurons,APOE alleles differentially influenced endogenous DNA damage,DNA repair,and neuronal motility. Single‐cell RNA sequencing revealed APOE4‐specific gene expression signatures associated with AD,whereas APOE2 GABAergic neurons were enriched for DNA repair and signaling pathways. Consistent with this,APOE2 neurons exhibited significantly lower levels of DNA damage. APOE4 GABAergic neurons exhibit increased expression of repetitive ribosomal RNA,which is associated with DNA damage and cellular senescence. To determine whether the effects extended to excitatory neurons,we used a separate human model of Ngn2‐induced glutamatergic neurons,and found that APOE2 excitatory neurons were more resistant to cellular senescence and DNA damage than isogenic APOE3 and APOE4 neurons. Similarly,we found human APOE2‐targeted replacement mice exhibited less nucleolar enlargement and increased nuclear Lamin A/C,Hmgb1,and H3K9me3 compared to APOE4 counterparts. Together,our findings identify DNA repair and suppression of senescence‐associated processes as key mechanisms by which APOE2 is associated with neuronal resilience,providing mechanistic insight into its association with exceptional longevity and protection against AD. Neurons expressing APOE2 were more resistant to endogenous DNA damage,activated transcriptional signaling pathways associated with DNA repair,and were resilient to stress‐induced DNA damage and cellular senescence. In contrast,APOE4 neurons exhibited elevated expression of rRNA repetitive elements and were prone to becoming senescent. View Publication

Background: Diffuse Midline Glioma H3K27-altered (DMG) is an extremely aggressive and lethal childhood brain cancer that grows within the midline structure of the brain. Current treatment options are only palliative,making DMG in desperate need for therapeutic breakthroughs. One of the major challenges limiting the study of DMG is the lack of reliable preclinical models. In-vivo mouse models are expensive and technically challenging and in-vitro cell culture models lack the essential components of tumor microenvironment (TME) needed to recapitulate the complex biology of these tumors. Scalable human planar neural organoids (PNOs) with multi-cellular make-up can serve as a cost effective and reliable model system to capture DMG biology and allow effective species matched drug testing in-vitro. Methods: Using 3 separate DMG patient derived xenograft (PDX) cell lines,we spatially profiled a novel scalable human iPSC-derived PNO system containing neurons,functional astrocytes and microglia using the NanoString GeoMx spatial transcriptomics system. Results: We found that all three cell lines interact with and integrate into the human PNOs,demonstrating favorable growth conditions in a complex co-culture. Across spatially resolved regions of interest (ROI’s) tumor cells individually interact with microglia and astrocytes and transcriptomic profiling of these mixed cell ROI’s shows differences in the genetic signatures of both the normal cells (microglia/astrocytes) and the tumor cells. When compared to biopsies obtained directly from DMG patients,DMG cells within PNOs correlate strongly at both transcriptomic and proteomic levels. The multi-cellular PNOs also enabled drug target bystander toxicity screening not possible in a traditional tumor cell only monoculture. Conclusion: This study provides a proof-of-concept for scalable PNO modeling for DMG and underscores the translational relevance of this model system. 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

Diverse risk genes have been identified for neurodevelopmental disorders (NDDs),but how these genes converge on similar biological pathways in neurons,and thus give rise to similar phenotypes,is unclear. Here we apply a pooled CRISPR approach to successfully target 23 NDD loss-of-function genes with roles in chromatin biology and examine convergent effects on gene expression across human induced pluripotent stem cell-derived neural progenitor cells,glutamatergic neurons and GABAergic neurons. Points of convergence vary between these cell types,with the greatest number of convergent genes and strongest convergent networks in mature glutamatergic neurons,where they broadly represent synaptic,epigenetic and,unexpectedly,mitochondrial pathways. The most convergent networks were observed between NDD genes with shared biological annotations,clinical associations and co-expression patterns in human post-mortem brain. Drugs that were predicted to reverse convergent transcriptomic signatures and/or arousal and sensory processing behaviors ameliorated behavioral phenotypes in zebrafish NDD gene mutants. These results suggest that convergent effects of NDD risk genes could provide clinically useful insights. By studying 23 neurodevelopmental disorder genes across model systems and brain cell types,the authors uncovered shared downstream effects that converge on synaptic biology,epigenetic regulation and mitochondrial function. View Publication

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

Human organoids are considered physiological models that reflect human physiology; however,their applications in drug screening studies are limited. To develop a fundamental treatment for recurrent Crohn’s disease (CD),in which tumor necrosis factor (TNF) is a key pathogenic factor,we conducted phenotypic drug screening to prevent TNF-induced cell death in human intestinal epithelial organoids. Glycyrrhizin,a natural product of licorice root,dose-dependently blocked TNF-induced cell death in organoids but not in TNF-sensitive L929 cells; L929 cells exhibited necroptosis,whereas organoid-derived cells preferentially showed apoptosis upon TNF treatment,determining the specificity of glycyrrhizin. Glycyrrhizin inhibited downstream caspase-8 signaling,which is essential for TNF-dependent apoptosis,and ameliorated intestinal inflammation in vivo. These results demonstrate that glycyrrhizin may be a novel therapeutic compound for CD and highlight the importance of using organoids for phenotypic drug screening. Graphical abstract Highlights•Human intestinal organoids are sensitive to TNF-induced cytotoxicity•We developed an assay system to screen for compounds that resist TNF in organoids•Glycyrrhizin inhibited TNF-induced apoptosis but not necroptosis in organoids•Glycyrrhizin ameliorated intestinal inflammation in a murine model in vivo Takahashi et al. developed a high-throughput phenotypic screening platform using human intestinal organoids,which exhibited higher sensitivity to TNF than conventional intestinal epithelial cell lines,and identified glycyrrhizin that protected TNF-induced cell death both in organoids and in vivo. Phenotypic screening using organoids,as demonstrated in this study,paves the way for drug development and chemical biology. View Publication

Introduction: Extensive thermal injury remains a formidable clinical challenge,primarily due to the profound deficit of autologous donor skin,which necessitates prolonged hospitalization and escalates healthcare expenditures. While human embryonic stem cells (hESCs) offer a theoretically inexhaustible source for regenerative therapy,optimizing their differentiation and engraftment remains critical for clinical translation. Methods: We used a three-stage protocol to induce the differentiation of hESCs into keratinocytes (KCs). To optimize the delivery of hESC-derived keratinocytes (EKCs),human dermal fibroblasts (HFBs) were utilized to provide essential extracellular matrix (ECM) and microenvironmental support. The two cell types could self-assemble into 3D spheroids. After optimizing the size and cell proportion,these spheroids were subsequently transplanted onto full-thickness dorsal wounds in immunodeficient mice to evaluate their regenerative capacity. Results: hESC-derived keratinocytes exhibited the expression of stage-specific epidermal markers,confirming high differentiation efficiency. In vitro,EKCs demonstrate the capacity to form stratified epidermal structures. By self-assembling into spheres with dermal fibroblasts,the EKCs demonstrated successful engraftment and sustained survival in vivo. The transplantation of these 3D spheroids significantly accelerated wound closure and re-epithelialization compared with controls. Conclusions: This study establishes a robust cell therapy approach characterized by a short preparation cycle with high differentiation efficiency and high transplantation survival rate,offering a novel strategy for the treatment of extensive skin defects. View Publication

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation,development,and utilization. However,the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore,generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study,we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos,using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro,exhibiting high expression of key pluripotency markers,genomic stability,and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. View Publication