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BackgroundHIV-1-associated neurocognitive impairment (HIV-1-NCI) is marked by ongoing and chronic neuroinflammation with loss and decline in neuronal function even when antiretroviral drug therapy (ART) successfully suppresses viral replication. Microglia,the primary reservoirs of HIV-1 in the central nervous system (CNS),play a significant role in maintaining this neuroinflammatory state. However,understanding how chronic neuroinflammation is generated and sustained by HIV-1,or impacted by ART,is difficult due to limited access to human CNS tissue.MethodsWe generated an in vitro model of admixed hematopoietic progenitor cell (HPC) derived microglia embedded into embryonic stem cell (ESC) derived Brain Organoids (BO). Microglia were infected with HIV-1 prior to co-culture. Infected microglia were co-cultured with brain organoids BOs to infiltrate the BOs and establish a model for HIV-1 infection,“HIV-1 M-BO”. HIV-1 M-BOs were treated with ART for variable directions. HIV-1 infection was monitored with p24 ELISA and by digital droplet PCR (ddPCR). Inflammation was measured by cytokine or p-NF-kB levels using multiplex ELISA,flow cytometry and confocal microscopy.ResultsHIV-1 infected microglia could be co-cultured with BOs to create a model for “brain” HIV-1 infection. Although HIV-1 infected microglia were the initial source of pro-inflammatory cytokines,astrocytes,neurons and neural stem cells also had increased p-NF-kB levels,along with elevated CCL2 levels in the supernatant of HIV-1 M-BOs compared to Uninfected M-BOs. ART suppressed the virus to levels below the limit of detection but did not decrease neuroinflammation.ConclusionsThese findings indicate that HIV-1 infected microglia are pro-inflammatory. Although ART significantly suppressed HIV-1 levels,neuronal inflammation persisted in ART-treated HIV-1 M-BOs. Together,these findings indicate that HIV-1 infection of microglia infiltrated into BOs provides a robust in vitro model to understand the impact of HIV-1 and ART on neuroinflammation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03375-w. View Publication

SummaryKCNQ1/Kv7,a low-voltage-gated K+ channel,regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes,its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s),we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation,but affected channel function by increasing spike frequency and Ca2+ flux,leading to insulin hypersecretion. With prolonged culturing,the mutant islets decreased their secretion and gradually deteriorated,modeling a diabetic state,which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and ?-cell survival in hereditary diabetes pathology. Graphical abstract Highlights•A permanent neonatal diabetes melitus patient carries a homozygous KCNQ1 mutation•KCNQ1R397W is loss of function and shows atypical electrophysiology in hESC-islets•Under high glucose,elevated Ca2+ flux leads to insulin hypersecretion•Mutant cells gradually switch phenotype,deteriorate,accelerated by high glucose Biological sciences; Endocrinology; Endocrinology; Health sciences; Internal medicine; Medical specialty; Medicine; Natural sciences; Physiology View Publication

Personalized antisense oligonucleotides (ASOs) have achieved positive results in the treatment of rare genetic disease1. As clinical sequencing technologies continue to advance,the ability to identify patients with rare disease harbouring pathogenic genetic variants amenable to this therapeutic strategy will probably improve. Here we describe a scalable platform for generating patient-derived cellular models and demonstrate that these personalized models can be used for preclinical evaluation of patient-specific ASOs. We describe protocols for delivery of ASOs to patient-derived organoid models and confirm reversal of disease-associated phenotypes in cardiac organoids derived from a patient with Duchenne muscular dystrophy (DMD) with a structural deletion in the gene encoding dystrophin (DMD) that is amenable to treatment with existing ASO therapeutics. Furthermore,we designed novel patient-specific ASOs for two additional patients with DMD (siblings) with a deep intronic variant in the DMD gene that gives rise to a novel splice acceptor site,incorporation of a cryptic exon and premature transcript termination. We showed that treatment of patient-derived cardiac organoids with patient-specific ASOs results in restoration of DMD expression and reversal of disease-associated phenotypes. The approach outlined here provides the foundation for an expedited path towards the design and preclinical evaluation of personalized ASO therapeutics for a broad range of rare diseases. A scalable platform for generating patient-specific organoids for testing personalized oligonucleotide therapeutics is described. View Publication

BackgroundThree common isoforms of the apolipoprotein E (APOE) gene - APOE2,APOE3,and APOE4 - hold varying significance in Alzheimer’s Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer’s Disease (AD),and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely,APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However,it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB.MethodsTo explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta,we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey’s post hoc testing as appropriate.ResultsIsogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance,tight junction integrity and efflux transporter gene expression. However,recombinant APOE4 protein significantly impeded the “brain-to-blood” amyloid beta 1–40 (A?40) transport capabilities of BMEC-like cells,suggesting a role in diminished amyloid clearance. Conversely,APOE2 increased amyloid beta 1–42 (A?42) transport in the model. Furthermore,we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways,mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes,yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition,while APOE2 pericyte-like cells displayed the least amyloid deposition,an observation in line with vascular pathologies in AD patients.ConclusionsWhile APOE genotype did not directly impact general BMEC or pericyte properties,APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely,APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB,motivating further development of such in vitro models in AD modeling and drug development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00580-2. View Publication

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically,4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1),thereby mitigating oxidative stress. This has important relevance for cancer,as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress,thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically,high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells. How cells adapt to glucose starvation is still elusive. Here,Levy et al. show that the mTOR substrate 4EBP1 protects human,mouse,and yeast cells from glucose starvation and is exploited by cancer cells to promote tumorigenesis. View Publication

ABSTRACTMedium chain acyl?CoA dehydrogenase deficiency (MCADD) is an inherited metabolic disease,characterized by biallelic variants in the ACADM gene. Interestingly,even with the same genotype,patients often present with very heterogeneous symptoms,ranging from fully asymptomatic to life?threatening hypoketotic hypoglycemia. The mechanisms underlying this heterogeneity remain unclear. Therefore,there is a need for in vitro models of MCADD that recapitulate the clinical phenotype as a tool to study the pathophysiology of the disease. Fibroblasts of control and symptomatic MCADD patients with the c.985A>G (p.K329E) were reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs were then differentiated into hepatic expandable organoids (EHOs),further matured to Mat?EHOs,and functionally characterized. EHOs and Mat?EHOs performed typical hepatic metabolic functions,such as albumin and urea production. The organoids metabolized fatty acids,as confirmed by acyl?carnitine profiling and high?resolution respirometry. MCAD protein was fully ablated in MCADD organoids,in agreement with the instability of the mutated MCAD protein. MCADD organoids accumulated medium?chain acyl?carnitines,with a strongly elevated C8/C10 ratio,characteristic of the biochemical phenotype of the disease. Notably,C2 and C14 acyl?carnitines were found decreased in MCADD Mat?EHOs. Finally,MCADD organoids exhibited differential expression of genes involved in ??oxidation,mitochondrial ??oxidation,TCA cycle,and peroxisomal coenzyme A metabolism,particularly upregulation of NUDT7. iPSC?derived organoids of MCADD patients recapitulated the major biochemical phenotype of the disease. Mat?EHOs expressed relevant pathways involved in putative compensatory mechanisms,notably CoA metabolism and the TCA cycle. The upregulation of NUDT7 expression may play a role in preventing excessive accumulation of dicarboxylic acids in MCADD. This patient?specific hepatic organoid system is a promising platform to study the phenotypic heterogeneity between MCADD patients. View Publication

SummaryKnowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete,yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress,we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens,aggrecan,perlecan,proteoglycans,and elastic fibers. We identified two populations of chondroprogenitor cells,mesenchyme cells and nascent chondrocytes,and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage,but also play a pivotal autocrine cell signaling role in determining chondrocyte fate. Graphical abstract Highlights•Craniofacial cartilage organoids were grown from human neural crest stem cells•These organoids exhibited elastic cartilage architecture and characteristic markers•Paracrine signaling drove chondrogenesis in mesenchyme cells and nascent chondrocytes•ECM components cemented chondrocyte cell fate through autocrine signaling Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Specialized functions of cells View Publication

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage,yet the underlying regulatory mechanisms remain elusive. Here,we show that trophoblast specific deletion of Kat8,a MYST family histone acetyltransferase,leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs),we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker,CDX2,via acetylating H4K16. Remarkably,CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover,increasing H4K16ac via using deacetylase SIRT1 inhibitor,EX527,restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8,CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth,which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL,shedding light on early gestational abnormalities. Embryo implantation failure is a leading cause of miscarriage,though the mechanisms underlying trophoblast defects are not well understood. Here they show that the histone acetyltransferase KAT8 is essential for proper activation of the trophoblast stemness gene CDX2,and that placental development can be partially rescued by inhibiting histone deacetylase activity. View Publication

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3,SLMAP,and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states. View Publication

HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present,HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here,we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap,5?UTR,modified nucleotide composition,coding sequence optimization and poly(A) length,and their effects on mRNA translation over time. Importantly,using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest,highlighting the importance of screening methods tailored to the protein of interest,and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics. View Publication