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This study describes novel interactors of the retinal Crumbs complex and reveals homo- and heterotypic interactions of CRB1 and CRB2 that are not significantly affected by patient-associated mutations. Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis,which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1,and in zebrafish,Crb2 has been shown to interact through the extracellular domain. Here,we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs,CRB1 was enriched in CRB2 samples,supporting a CRB1–CRB2 interaction. Furthermore,novel interactors of the crumbs complex were identified,representing a retina-derived protein interaction network. Using co-immunoprecipitation,we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2,but not with CRB3,which lacks an extracellular domain. Next,we explored how missense mutations in the extracellular domain affect CRB1–CRB2 interactions. We observed no or a mild loss of CRB1–CRB2 interaction,when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together,our results show a stable interaction of human canonical CRB2 and CRB1 in the retina. View Publication

Ribosomal RNA (rRNA) genes exist in multiple copies arranged in tandem arrays known as ribosomal DNA (rDNA). The total number of gene copies is variable,and the mechanisms buffering this copy number variation remain unresolved. We surveyed the number,distribution,and activity of rDNA arrays at the level of individual chromosomes across multiple human and primate genomes. Each individual possessed a unique fingerprint of copy number distribution and activity of rDNA arrays. In some cases,entire rDNA arrays were transcriptionally silent. Silent rDNA arrays showed reduced association with the nucleolus and decreased interchromosomal interactions,indicating that the nucleolar organizer function of rDNA depends on transcriptional activity. Methyl-sequencing of flow-sorted chromosomes,combined with long read sequencing,showed epigenetic modification of rDNA promoter and coding region by DNA methylation. Silent arrays were in a closed chromatin state,as indicated by the accessibility profiles derived from Fiber-seq. Removing DNA methylation restored the transcriptional activity of silent arrays. Array activity status remained stable through the iPS cell re-programming. Family trio analysis demonstrated that the inactive rDNA haplotype can be traced to one of the parental genomes,suggesting that the epigenetic state of rDNA arrays may be heritable. We propose that the dosage of rRNA genes is epigenetically regulated by DNA methylation,and these methylation patterns specify nucleolar organizer function and can propagate transgenerationally. View Publication

Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we describe long-term GLICOs,a novel model in which COs are grown from embryonic stem cell cultures containing low levels of GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion,and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However,as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes,which secreted various pro-tumorigenic ligands including FGF1,as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype,the close association between glioma aggressiveness and patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01755-6. View Publication

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A,we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3,HAAO,HNF1A,MAP3K11) and previously unidentified (ABCD3,CDKN2AIP,USH1C,VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs,the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1,GAD2 and HOPX gene expression,potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall,our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function,and set up a framework for gene discovery and functional validation. Here,the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells,and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant. View Publication

ABSTRACTGenetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study looked at people from 12 unrelated families with variants in the gene SMPD4,a neutral sphingomyelinase that metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These individuals have severe developmental brain malformations,including microcephaly and cerebellar hypoplasia. The disease mechanism of SMPD4 was not known and so we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia,a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells lacking SMPD4 exhibit neural progenitor cell death and have shortened primary cilia,which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development. Summary: Mouse and human stem cell models of SMPD4 loss of function demonstrate that SMPD4 promotes cilia function and neural development. View Publication

IntroductionRetinal ganglion cells (RGCs) are susceptible to degenerative conditions such as glaucoma and traumatic optic neuropathies,which lead to vision loss. MicroRNA-21-5p has demonstrated potential neuroprotective effects,but its mechanisms in optic nerve injury remain underexplored. This study evaluates the neuroprotective role of microRNA-21-5p derived from induced pluripotent stem cells (iPSCs) in an optic nerve crush (ONC) model.Materials and methodsIn vitro qPCR demonstrated that the expression of microRNA-21-5p was increased in the co-culture medium of RGCs and iPSCs. Subsequently,in the in vivo experiments,we used a microRNA-21-5p agonist to assess its protective effects on RGCs. RNA sequencing was then performed in a mouse ONC model after treatment with a microRNA-21-5p agonist to explore the mechanisms underlying its neuroprotective effects on RGCs.ResultsAs demonstrated in our previous experiments,the RGCs-iPSCs co-culture group led to a higher survival rate of RGCs,as indicated by live/dead cell staining,compared to the RGCs-only group. Quantitative PCR (qPCR) results revealed a significant increase in the expression of microRNA-21-5p in the medium of the RGCs-iPSCs co-culture group. Furthermore,the survival rate of mouse retinal RGCs treated with a microRNA-21-5p agonist was significantly greater than that of the control group. Lastly,RNA sequencing of the retina from microRNA-21-5p agonist-treated mice indicated that microRNA-21-5p plays a protective role in RGCs by downregulating the expression of several genes,including Irf1,Ccl4,Itk,Cxcr2,Dclre1c,Traf1,Traf2,Rbl1,Cxcl5,Cxcl3,Cxcl1,Cxcl9,Il2rg,Cd3e,Cd3d,Cxcl10,Ccl5,Ccl12,Tap1,and Cxcr4.ConclusionMicroRNA-21-5p derived from iPSCs can enhance the survival rate of RGCs in the ONC model. This suggests that microRNA-21-5p may represent a novel and effective strategy for repairing RGC damage. Such a strategy could potentially be realized through the modulation of apoptosis,T-cell regulatory pathways,or TNF-? signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12886-025-04244-z. View Publication

BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass,irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications,neurodegeneration,and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity,homeostasis,and repair. Here,we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome,their modulation,and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ? 5 Gy) were treated with iMSC CM,endothelial cell viability,adherence,spheroid compactness,and proangiogenic sprout formation were significantly ameliorated,and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently,iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore,iMSC CM suppressed IR-induced NF?B activation,TNF-? release,and ROS production in THP1 cells. Additionally,iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3,iMSCs,or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2,IL6,IL8/CXCL8,ANG (Angiogenin),GRO?/CXCL1,and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors,the PI3K/AKT/mTOR modulator PRAS40 and ?-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism,translation,mitochondrial respiration,DNA damage repair,and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03847-5. View Publication

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes,including SOD1. So far,clinical records,rodent studies,and in vitro models have yielded arguments for either a primary motor neuron disease,or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin,in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC,the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings,we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model,myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers,lack of sarcomeres,morphologically different nAChR clusters,and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably,trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary,a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS. View Publication

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies,the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner,making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN),complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2,with preclinical evidence to support their efficacy in T cell malignancies. The T cell receptor β-chain is expressed in two isoforms,TRBC1 and TRBC2,with clonally expanded mature T cell lymphomas expressing one of them exclusively,while healthy T cells randomly express either TRBC1 or TRBC2. Here authors show structure-based design of a TRBC2-specific antibody,and depletion of malignant T cells carrying TRBC1 or TRBC2 with CAR-T cells against the cognate receptor chain in murine models. View Publication

IntroductionBAP1 is a deubiquitinase (DUB) of the Ubiquitin C-terminal Hydrolase (UCH) family that regulates gene expression and other cellular processes,through its direct catalytic activity on the repressive epigenetic mark histone H2AK119ub,as well as on several other substrates. BAP1 is also a highly important tumor suppressor,expressed and functional across many cell types and tissues. In recent work,we demonstrated a cell intrinsic role of BAP1 in the B cell lineage development in murine bone marrow,however the role of BAP1 in the regulation of B cell mediated humoral immune response has not been previously explored. Methods and resultsIn the current study,we demonstrate that a B-cell intrinsic loss of BAP1 in activated B cells in the Bap1 fl/fl Cγ1-cre murine model results in a severe defect in antibody production,with altered dynamics of germinal centre B cell,memory B cell,and plasma cell numbers. At the cellular and molecular level,BAP1 was dispensable for B cell immunoglobulin class switching but resulted in an impaired proliferation of activated B cells,with genome-wide dysregulation in histone H2AK119ub levels and gene expression. Conclusion and discussionIn summary,our study establishes the B-cell intrinsic role of BAP1 in antibody mediated immune response and indicates its central role in the regulation of the genome-wide landscapes of histone H2AK119ub and downstream transcriptional programs of B cell activation and humoral immunity. View Publication

SummaryFollicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin,which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies,combined with machine-learning approaches,we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed,proliferative,and chromatin-modifying states,with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling,we find that each state was associated with a combination of mutations in chromatin modifiers,copy-number alterations to TNFAIP3,and T follicular helper cells (Tfh) cell interactions,or primarily by a microenvironment rich in activated T cells. Altogether,these data define FL B cell transcriptional states across a large cohort of patients,contribute to our understanding of FL heterogeneity at the tumor cell level,and provide a foundation for guiding therapeutic intervention. Graphical abstract Highlights•B cells from follicular lymphoma exhibit 3 distinct transcriptional states•FL B cells differ by enhanced inflammation,proliferation,or chromatin remodeling•Tumor cell states correlate with unique immune-microenvironment features•Unique mutation and CNV profiles highlight potential genetic causes of heterogeneity Krull et al. analyzed bulk transcriptional,genomic,and immune profiles of B cells from follicular lymphoma and reveal 3 distinct transcriptional states. These cell states underscore the inherent variability of FL tumors,independent of stroma,and implicate intrinsic differences as an underpinning to FL heterogeneity. View Publication

AbstractBacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology,including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis,suggesting there is a defect in apoptotic clearance. In this study,we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK,TYRO3,AXL,integrin αVβ5,CD36,and TIM-3,whereas TIM-1,αVβ3,CD300b,CD300f,STABILIN-1,and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant,suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release,indicating effective protease inhibition,and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3. View Publication