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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

SummaryThe medial ganglionic eminence (MGE) gives rise to parvalbumin (PV)- and somatostatin (SST)-expressing cortical interneurons essential for regulating cortical excitability. Although PV interneurons are linked to various neurodevelopmental and neurodegenerative disorders,reliably generating them from human pluripotent stem cells (hPSCs) has been extremely challenging. We present a robust,reproducible protocol for generating single-rosette MGE organoids (MGEOs) from hPSCs. Transcriptomic analyses reveal that MGEOs exhibit MGE regional identity and faithfully model the developing human fetal MGE. As MGEOs mature,they generate abundant PV-expressing cortical interneurons,including putative basket and axoaxonic cells,at a scale not previously achieved in vitro. When fused with hPSC-derived cortical organoids,these interneurons rapidly migrate into cortical regions,integrate into excitatory networks,and contribute to complex electrophysiological patterns and the emergence of large numbers of fast-spiking neurons. MGEOs thus offer a powerful in vitro approach for probing human MGE-lineage cortical and subcortical GABAergic neuron development,modeling various neuropsychiatric disorders,and advancing cell-based therapies for neurodevelopmental and neurodegenerative disorders. Graphical abstract View Publication

DNA methylation regulation involves multi-layered chromatin interactions that require remodeling proteins like the helicase,lymphoid-specific (HELLS). Here,we generate HELLS and DNA methyltransferase 3A and B (DNMT3A/B) knockout human pluripotent stem cells and report telomere-to-telomere maps of whole genome bisulfite sequencing data combined with ATAC-sequencing. Disrupting HELLS induces a global loss of DNA methylation that is distinct from the DNMTs,in particular over peri/centromeric satellite repeats as defined in the telomere-to-telomere genome assembly. However,HELLS appears dispensable for local enhancer remodeling and the potential to differentiate into the three embryonic germ layers. Taken together,our results further clarify the genomic targets and role of HELLS in human cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03681-9. 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

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

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

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

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

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

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

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

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