技术资料

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes,but its efficacy is hindered by low oxygen supply post-transplantation,particularly in subcutaneous spaces and encapsulation devices,leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here,we show that ? cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1,FOS,and JUN),which downregulates key ? cell transcription factors. We further identified genes important for maintaining ? cell fitness in hypoxia,with EDN3 as a potent player. Elevated EDN3 expression preserves ? cell identity and function in hypoxia by modulating genes involved in ? cell maturation,glucose sensing and regulation. These insights improve the understanding of hypoxia’s impact on stem cell-derived islets,offering a potential intervention for clinical applications. Hypoxia impairs the efficacy of stem cell-derived islet cell therapy,making it a potential barrier for treatment of type 1 diabetes. Wang et al. identify EDN3 as a key factor that preserves ? cell identity and function in hypoxia,offering possible strategies to improve therapeutic outcomes. View Publication

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here,to understand the pathological mechanisms of Kd,we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis,astrogenesis,and oligodendrogenesis but manifest early myelination defects. Specifically,Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly,myelin is affected in the absence of autophagy and mTOR pathway dysregulation,suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions,Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding,Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether,this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd. View Publication

Interactions between adipose tissue,liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model,we establish an interconnected microphysiological system (MPS) containing white adipocytes,hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS,we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function,whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance,corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases. In vitro modelling of the adipose tissue-liver axis can advance understanding and therapy of metabolic disease,including by distinguishing effects of obesity and inflammation. Here,authors develop such a system based on isogenic human iPSCs and interconnected microphysiological devices. View Publication

In this study,we developed a cell system to study TCF4 in human oligodendrocyte differentiation,showed that TCF4 regulates human oligodendroglial differentiation in a dose-dependent manner,and established a system to dissect TCF4 function in a human tissue–like context. Heterozygous mutations of TCF4 in humans cause Pitt–Hopkins syndrome,a neurodevelopmental disease associated with intellectual disability and brain malformations. Although most studies focus on the role of TCF4 in neural stem cells and neurons,we here set out to assess the implication of TCF4 for oligodendroglial differentiation. We discovered that both monoallelic and biallelic mutations in TCF4 result in a diminished capacity to differentiate human neural progenitor cells toward myelinating oligodendrocytes through the forced expression of the transcription factors SOX10,OLIG2,and NKX6.2. Using this experimental strategy,we established a novel organoid model,which generates oligodendroglial cells within a human neurogenic tissue–like context. Also,here we found a reduced ability of TCF4 heterozygous cells to differentiate toward oligodendroglial cells. In sum,we establish a role of human TCF4 in oligodendrocyte differentiation and provide a model system,which allows to dissect the disease etiology in a human tissue–like context. View Publication

The neuromuscular junction (NMJ) is essential for transmitting signals from motor neurons (MNs) to skeletal muscles (SKMs),and its dysfunction can lead to severe motor disorders. However,our understanding of the NMJ is limited by the absence of accurate human models. Although human induced pluripotent stem cell (iPSC)-derived models have advanced NMJ research,their application is constrained by challenges such as limited differentiation efficiency,lengthy generation times,and cryopreservation difficulties. To overcome these limitations,we developed a rapid human NMJ model using cryopreserved MNs and SKMs derived from iPSCs. Within 12 days of coculture,we successfully recreated NMJ-specific connectivity that closely mirrors in vivo synapse formation. Using this model,we investigated amyotrophic lateral sclerosis (ALS) and replicated ALS-specific NMJ cytopathies with SOD1 mutant and corrected isogenic iPSC lines. Quantitative analysis of 3D confocal microscopy images revealed a critical role of MNs in initiating ALS-related NMJ cytopathies,characterized by alterations in the volume,number,intensity,and distribution of acetylcholine receptors,ultimately leading to impaired muscle contractions. Our rapid and precise in vitro NMJ model offers significant potential for advancing research on NMJ physiology and pathology,as well as for developing treatments for NMJ-related diseases. View Publication

BackgroundGenome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool,but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore,genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations,but not in single cells. However,systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells,it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.MethodsIn this study,we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps,which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.ResultsWe analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations,including insertions and deletions,such as 1-bp insertions and 8-bp deletions. Moreover,we successfully cryopreserved and then recovered genome-edited iPS cell clones,demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.ConclusionsThis study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04414-2. View Publication

Quantification of subcellular structures such as nuclei and cytoplasmic proteins using staining methods based on fluorescent dyes or fluorescently tagged antibodies are widely used in scientific research. Accurate high-throughput quantitation of these assays can be time consuming and challenging. Here,we present our FIJI based Semi-Automated counting Macro termed SAM,and we validate its accuracy against manual counting and other automated counting methods. By introducing this automated quantification tool,we aim to contribute to the ongoing efforts to enhance the reliability,efficiency,and standardization of immunostaining analysis in the field of diabetes research and beyond.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12144-x. View Publication

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

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals,we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs),which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency,the NANOG-OCT4 Complex,and its associated network are significantly enriched for regulatory variants with large effects,suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks,shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity,and suggests that genetic factors may contribute to cell state transitions in human iPSC lines. Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Here,authors show that pluripotency and self-renewal processes have a high level of regulatory complexity and suggest that genetic factors contribute to cell state transitions in human iPSC lines. 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