技术资料

Mesenchymal stromal cells (MSCs) have shown immunomodulatory effects and great promise in many inflammatory diseases such as acute respiratory distress syndrome (ARDS). However,several barriers to translation remain such as cell availability and potency. This study evaluates the therapeutic potentials of three types of MSCs,bone marrow-derived MSCs (BM-MSC),the human induced pluripotent stem cell-derived MSC wild type (iMSC WT) and β2 microglobulin-knockout iMSCs (iMSC B2M KO) with or without proinflammatory cytokine preconditioning. BM-MSC,iMSC WT and iMSC B2M KO were preconditioned with a proinflammatory cytokine cocktail (Cytomix: IL-1β,IFN-γ and TNF-α). Immunoregulatory biomarkers were analysed by flow cytometry and cytokines released by ELISA. MSC antimicrobial properties were analysed via CFU assays while the MSCs’ immunomodulatory effects were evaluated using macrophage activation and T cell proliferation assays. Proinflammatory cytokine preconditioning enhanced the therapeutic potency of all three types of MSCs by increasing immunomodulatory marker expression,enhancing the antimicrobial effects and improving MSC-mediated inhibition of T cell proliferation. These findings provided new insights into the therapeutic potencies of MSCs in inflammation. Further studies are required for in vitro characterisation of the MSCs and in vivo efficacy verification of these MSCs prior to their clinical application. View Publication

Huntington’s disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression,providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However,achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here,we developed allele-selective,CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD,spinocerebellar ataxias types 1 and 3,and dentatorubral pallidoluysian atrophy. The most promising candidate,amiR136-13A,reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover,amiR136-13A reduced the level of HTT1a,a short pathogenic isoform of huntingtin. Collectively,these findings identify amiR136-13A as a potent,selective,and safe therapeutic candidate for HD and potentially other polyQ disorders. View Publication

Widespread uses of nuclear materials increase the risk of accidental or intentional radiation exposure,which can result in acute radiation syndrome (ARS). Hematopoietic ARS (H-ARS) occurs at relatively low doses and is potentially lethal without intervention. While several FDA-approved cytokine-based radiomitigators exist,many require repeated dosing,complicating deployment in mass-casualty scenarios. This study evaluated a novel long-acting,murine-reactive granulocyte–macrophage colony-stimulating factor (LA-GM-CSF; mPDM608) as a prophylactic and mitigative countermeasure for H-ARS. Male and female C57BL/6 mice were exposed to lethal or sublethal total body irradiation (TBI) and treated with LA-GM-CSF using single- or multi-dose regimens administered before or after TBI. Safety,30-day survival,hematologic recovery,bone marrow cellularity,serum GM-CSF pharmacokinetics,endothelial injury markers,and cytokine profiles were assessed using standard hematology,histopathology,ELISA,and multiplex assays. LA-GM-CSF was well tolerated at doses up to 30 mg/kg. Single or limited dosing conferred significant survival benefits compared with vehicle controls,with optimal efficacy observed at lower doses (3 mg/kg). Post-TBI administration as a single dose 24 h after exposure markedly improved survival in both sexes,with stronger hematopoietic recovery in males. LA-GM-CSF accelerated recovery of neutrophils,red blood cells,platelets,hematocrit,and sternal megakaryocytes,prolonged circulating GM-CSF levels,and favorably modulated endothelial injury markers and select cytokines. LA-GM-CSF demonstrates strong potential as a next-generation radiation countermeasure,providing robust survival benefit and hematopoietic recovery with minimal dosing. The results shown here support further development for H-ARS management under the FDA Animal Rule. View Publication

Background/Objectives: Overexpression of transferrin receptor (TFR1) is common in cancer and may be associated with inferior treatment outcomes. Due to these patterns and TFR1’s essential role in iron metabolism,the protein has been targeted for cytotoxic drug delivery. More recently,increased TFR1 expression has been linked to tumor microenvironment (TME) infiltration by immune effectors in selected tumors,but a comprehensive assessment of the genomic landscape associated TFRC (the gene encoding TFR1) expression has not been conducted. Methods: By utilizing a pan-cancer database of 93,248 patients with whole-exome and whole-transcriptome sequencing,we assessed TFRC-associated multiomic patterns. Results: We found that high TFRC expression correlates with significantly worse overall survival in multiple common solid tumor types,a higher tumor mutational burden (TMB),an increase in infiltrating effector cells with upregulated immune checkpoint markers within the TME,and increased frequency of specific high-risk genomic alterations. Further assessment in cell line models revealed increased susceptibility to cytotoxic T cells when iron metabolism is elevated,despite upregulation of the checkpoint ligand PD-L1. Conclusions: High TFRC expression,therefore,indicates worse clinical risk across multiple common tumor types but potentially increased susceptibility to cytotoxic immune effectors,informing the development of TFR1 biomarker-driven therapeutic strategies. 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

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

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

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

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

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

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

Background: Acute erythroid leukemia (AEL) or AML-M6 predominantly affects older adults and is rare in childhood. Compared with other AML subtypes,AEL remains relatively understudied because of its rarity. We established LS-CHM,a novel AEL cell line derived from the ascitic fluid of a patient with congenital leukemia. Interestingly,leukemic cells persisted in the ascitic fluid even after successful eradication from the bone marrow and extramedullary sites. Method: Leukemia cells from the ascites fluid exhibited robust proliferation in culture independent of cytokine requirement and were further characterized by flow cytometric immunophenotyping,cytogenetics,cell cycle and doubling time analysis,colony formation,genome and RNA sequencing,myeloid gene next generation sequencing,and cytotoxicity analysis. Results: LS-CHM displayed CD36,partial CD235a,CD31,CD43,and CD71 expression and demonstrated in vitro robust growth and high sensitivity to chemotherapeutic agents. A PDX mouse model showed development of leukemia. Genomic analysis revealed a frameshift BCOR mutation in the absence of additional mutations and downregulated TP53 expression with an exonic non-deleterious mutation. RNA sequencing of LS-CHM cells revealed upregulation of two cohesin complex genes,RAD21 and SMC3,whose high levels are associated with hematopoietic stem cell differentiation into erythroid lineage. Conclusions: LS-CHM represents the first congenital AEL-derived cell line,in contrast to the predominantly adult-origin and often secondary erythroid leukemia cell lines available currently. Thus,LS-CHM provides a unique pediatric and extramedullary AEL model,expanding the existing spectrum of AEL cell lines and offering valuable opportunities for biologic and therapeutic investigations. View Publication