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Seven female individuals with multiple congenital anomalies,developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T,p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined,and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways,we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling,transcriptomics and protein analysis,comparing healthy and variant cells. When comparing the two cell lines,we identified altered gene expression in neural cells expressing the variant,including genes regulating RNA polymerase II activity,transcription,pre-mRNA processing,and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development,forebrain differentiation,and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model,expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5. View Publication

IntroductionExposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species,along with the financial and ethical challenges associated with animal testing,the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously,we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay,ReproTracker,that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.MethodsTo further increase the predictability of the assay in identifying potential teratogens,we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens,including the compounds listed in the ICH S5 reference list.ResultsThe optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall,based on the current dataset,the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%),when compared to the previously described assay.DiscussionIn summary,trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker,making the assay an invaluable tool for early in vitro teratogenicity screening. View Publication

Prion diseases,such as sporadic Creutzfeldt-Jakob Disease (sCJD),are neurodegenerative disorders caused by misfolding of the prion protein (PrP). The D178N mutation in the PrP gene causes Fatal Familial Insomnia (FFI). Here we show that both sCJD and FFI prions can infect human cerebral organoids with or without the D178N mutation,and that the resulting infection is dictated by the inoculating prion and not the host organoid genotype. View Publication

Biomolecular condensates are found throughout a diversity of eukaryotic cell types and cellular compartments,playing roles in various cellular functions. A given protein generally forms functionally and compositionally heterogeneous condensates,but the underlying regulatory mechanisms are unknown. Here,we found that different RNA motifs modulate the formation of heterogeneous mRNA-protein condensates via riboregulation. Fragile X-related 1 (FXR1),an RNA-binding protein interacting with nuclear pores,assembles distinct localized subcellular mRNP condensates linked to cytosolic accumulation of G-quadruplex-containing pluripotent mRNAs and the localized translation of nucleoporin mRNAs at nuclear pores. The diverse locations of FXR1 condensates depend on the unique RNA-protein interaction modules of its two RNA binding domains,and the opposing effects of different RNA motifs on the affinity of FXR1 for nuclear pores. Notably,reduced FXR1 levels and impaired nuclear pore function lead to the nuclear accumulation of transcribed RNAs,facilitating fate transition in human embryonic stem cells. Preventing this decline would result in impaired hESC differentiation. Subject terms: RNA metabolism,Embryonic stem cells,RNA,RNA transport View Publication

Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them,3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However,primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues,recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study,we developed a protocol to differentiate high-purity skin cells,such as fibroblasts (hFIBROs) and keratinocytes (hKERAs),from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE),a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently,hKERAs were seeded onto the dermis,and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover,exposure to Triton X-100,a known skin irritant,led to marked epidermal damage and significantly reduced cell viability,validating the model’s functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications,including the replacement of animal testing. View Publication

The primary cilium is a signal transduction organelle whose dysfunction clinically causes ciliopathies in humans. RAB23 is a small GTPase known to regulate the Hedgehog signalling pathway and ciliary trafficking. Mutations of RAB23 in humans lead to Carpenter syndrome (CS),an autosomal recessive disorder clinically characterized by craniosynostosis,polysyndactyly,skeletal defects,obesity,and intellectual disability. Although the clinical features of CS bear some resemblance to those of ciliopathies,the exact relationship between the pathological manifestations of CS and the ciliary function of RAB23 remains ambiguous. Besides,the in vivo ciliary functions of RAB23 remain poorly characterised. Here,we demonstrate in vivo and in vitro Rab23 loss-of-function mutants modelling CS,including Rab23 conditional knockout (CKO) mouse mutants,CS patient-derived induced pluripotent stem cells (iPSCs),and zebrafish morphants. The Rab23-CKO mutants exhibit multiple developmental and phenotypical traits recapitulating the clinical features of human ciliopathies and CS,indicating a causal link between the loss of Rab23 and ciliopathy. In line with the ciliopathy-like phenotypes,all three different vertebrate mutant models consistently show a perturbation of primary cilia formation,intriguingly,in a context-dependent manner. Rab23-CKO mutants reveal cell-type specific ciliary abnormalities in chondrocytes,mouse embryonic fibroblasts,neural progenitor cells and neocortical neurons,but not in epithelial cells,cerebellar granule cells and hippocampus neurons. A profound reduction in ciliation frequency was observed specifically in neurons differentiated from CS patient iPSCs,whereas the patients’ fibroblasts,iPSCs and neural progenitor cells maintained normal ciliation percentages but shortened cilia length. Furthermore,Rab23-KO neural progenitor cells show perturbed ciliation and desensitized to primary cilium-dependent activation of the Hedgehog signaling pathway. Collectively,these findings indicate that the absence of RAB23 causes dysfunctional primary cilia in a cell-type distinctive manner,which underlies the pathological manifestations of CS. Our findings present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. Through the use of patient-derived iPSCs differentiated cells,we present direct evidence of primary cilia anomalies in CS,thereby confirming CS as a ciliopathy disorder. Author summaryRAB23 mutations lead to Carpenter syndrome (CS),which manifests multiple clinical features resembling those of ciliopathies,a spectrum of disorders caused by defective primary cilia. However,the in vivo ciliary functions of RAB23 remain ambiguous. We established multiple Rab23 loss-of-function mutants,including conditional knockout (CKO) mouse mutants,CS patient-derived induced pluripotent stem cells (iPSCs),and zebrafish morphants. These mutant models consistently show context-dependent primary cilia anomalies. Rab23-CKO mutants display profound ciliary abnormalities in neocortical neurons,but not in epithelial cells,cerebellar granule cells and hippocampus neurons. Aberrant cilia formation and shortened cilia were observed in the neurons and neural progenitor cells derived from CS patient iPSCs. Furthermore,Rab23-KO neural progenitor cells exhibit impeded primary cilium-dependent Hedgehog signaling pathway transduction. Our findings suggest that the cell-type distinctive dysfunctional primary cilia may underlie the pathological manifestations of CS. We present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. The results from patient-derived iPSCs differentiated cells reveal direct evidence of primary cilia anomalies in CS,confirming CS as a ciliopathy disorder. View Publication

IntroductionAngelman Syndrome (AS) is characterized in large part by the loss of functional UBE3A protein in mature neurons. A majority of AS etiologies is linked to deletion of the maternal copy of the UBE3A gene and epigenetic silencing of the paternal copy. A common therapeutic strategy is to unsilence the intact paternal copy thereby restoring UBE3A levels. Identifying novel therapies has been aided by a UBE3A-YFP reporter mouse model. This study presents an analogous fluorescent UBE3A reporter system in human cells.MethodsPreviously derived induced Pluripotent Stem Cells (iPSCs) with a Class II large deletion at the UBE3A locus are used in this study. mGL and eGFP are integrated downstream of the endogenous UBE3A using CRISPR/Cas9. These reporter iPSCs are differentiated into 2D and 3D neural cultures to monitor long-term neuronal maturation. Green fluorescence dynamics are analyzed by immunostaining and flow cytometry.ResultsThe reporter is successfully integrated into the genome and reports paternal UBE3A expression. Fluorescence expression gradually reduces with UBE3A silencing in neurons as they mature. Expression patterns also reflect expected responses to molecules known to reactivate paternal UBE3A.DiscussionThis human-cell-based model can be used to screen novel therapeutic candidates,facilitate tracking of UBE3A expression in time and space,and study human-specific responses. However,its ability to restore UBE3A function cannot be studied using this model. Further research in human cells is needed to engineer systems with functional UBE3A to fully capture the therapeutic capabilities of novel candidates. View Publication

The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4,Sox2,Klf4,and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and have the potential to differentiate into any adult human cell type,offering a versatile tool for research and potential therapeutic applications. However,the efficiency of differentiation protocols for generating complex structures with multiple cell types,Like kidney organoids,remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using the stepwise 2D monolayer-based protocol developed by Morizane et al. 2017. We found that treating hiPSCs with 1–2% DMSO affects gene expression of pluripotent transcription factors,the epigenetic landscape,and hiPSC colony morphology. Our findings also suggest DMSO treatment enhances the expression of the key metanephric mesenchyme nephron progenitor marker,SIX2 after 9 days of kidney organoid differentiation and helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to fully elucidate the mechanisms underlying these effects and refine the differentiation process for potential in vitro research applications in biomedical research and drug development. View Publication

Degeneration of neuromuscular synapses is a key pathological feature of spinal muscular atrophy (SMA),yet cellular mechanisms underlying synapse dysfunction remain elusive. Here,we show that pharmacological stimulation with Roscovitine triggers the assembly of Munc13-1 release sites that relies on its local translation. Our findings show that presynaptic mRNA levels and local synthesis of Munc13-1 are diminished in motoneurons from SMA mice and hiPSC-derived motoneurons from SMA patients. Replacement of the Munc13-1 3’UTR with that of Synaptophysin1 rescues Munc13-1 mRNA transport in SMA motoneurons and restores the nanoscale architecture of presynaptic Munc13-1 release sites. Restoration of Munc13-1 levels leads to functional synaptic recovery in cultured SMA motoneurons. Furthermore,SMA mice cross-bred with a conditional knock-in mouse expressing modified Munc13-1 with a heterologous 3’UTR display attenuated synapse and neurodegeneration and improved motor function. Identifying Munc13-1 as an SMA modifier underscores the potential of targeting synapses to mitigate neuromuscular dysfunction in SMA. Defective neurotransmission is a hallmark of spinal muscular atrophy (SMA). Here,the authors show that local presynaptic Munc13-1synthesis is defective in SMA and that modification of the Munc13-1 mRNA rescues presynaptic architecture and excitability. View Publication

Disturbances within the cerebrovascular system substantially contribute to the pathogenesis of age-related cognitive impairment and Alzheimer’s disease (AD). Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-β (Aβ) in the leptomeningeal and cortical arteries and is highly prevalent in AD,affecting over 90% of cases. While the ε4 allele of apolipoprotein E ( APOE ) represents the strongest genetic risk factor for AD,it is also associated with cerebrovascular dysregulations. APOE plays a crucial role in brain lipid transport,particularly in the trafficking of cholesterol and phospholipids. Lipid metabolism is increasingly recognized as a critical factor in AD pathogenesis. However,the precise mechanism by which APOE influences cerebrovascular lipid signatures in AD brains remains unclear. In this study,we conducted non-targeted lipidomics on cerebral vessels isolated from the middle temporal cortex of 89 postmortem human AD brains,representing varying degrees of CAA and different APOE genotypes: APOE ε2/ε3 (N = 9),APOE ε2/ε4 (N = 14),APOE ε3/ε3 (N = 21),APOE ε3/ε4 (N = 23),and APOE ε4/ε4 (N = 22). Lipidomics detected 10 major lipid classes with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) being the most abundant lipid species. While we observed a positive association between age and total acyl-carnitine (CAR) levels (p = 0.0008),the levels of specific CAR subclasses were influenced by the APOE ε4 allele. Notably,APOE ε4 was associated with increased PE (p = 0.049) and decreased sphingomyelin (SM) levels (p = 0.028) in the cerebrovasculature. Furthermore,cerebrovascular Aβ40 and Aβ42 levels showed associations with sphingolipid levels including SM (p = 0.0079) and ceramide (CER) (p = 0.024). Weighted correlation network analysis revealed correlations between total tau and phosphorylated tau and lipid clusters enriched for PE plasmalogen and lysoglycerophospholipids. Taken together,our results suggest that cerebrovascular lipidomic profiles offer novel insights into the pathogenic mechanisms of AD,with specific lipid alterations potentially serving as biomarkers or therapeutic targets for AD. The online version contains supplementary material available at 10.1007/s00401-025-02949-5. View Publication

The increasing demand for efficient recombinant insulin production necessitates the development of scalable,high-yield,and cost-effective bioprocesses. In this study,we engineered a novel mini-proinsulin (nMPI) with enhanced expression properties by shortening the C-peptide and incorporating specific residue substitutions to eliminate the need for enzymatic cleavage. To optimize its production,we applied a hybrid approach combining microscale high-throughput cultivation using the BioLector microbioreactor and statistical modeling via response surface methodology (RSM). Critical medium components were first screened using Plackett–Burman Design (PBD) and refined through Central Composite Design (CDD),identifying glycerol as the most influential factor for yield. Among the four statistically derived formulations,Scenario III demonstrated the highest productivity in the microscale platform (13.00 g/L) and maintained strong performance upon scale-up to a 3-L bioreactor (11.5 g/L). The optimized medium balanced carbon and nitrogen sources to enhance cell viability and maximize protein expression. This study not only confirms the predictive accuracy and scalability of the hybrid optimization system but also introduces a robust production platform for nMPI that can be translated into industrial settings. The workflow presented here can serve as a model for the development of efficient expression systems for complex recombinant proteins in E. coli. View Publication

The CBFA2T3::GLIS2 (CG) fusion protein causes aggressive pediatric acute megakaryoblastic leukemia (AMKL). Although dysregulated molecular pathways in AMKL have been identified,their role in early pre-leukemic transformation remains poorly understood. We developed a disease model utilizing genetically modified human induced pluripotent stem cells (hiPSC) physiologically and conditionally expressing CG. Using in vitro differentiation and single-cell multi-omics,we captured the impact of oncogene activity on gene-regulatory networks during hematopoiesis. We discovered that CG interferes with myelopoiesis through two alternative routes: by locking aberrant megakaryocyte progenitors (aMKP) in a proliferative state,or by impeding differentiation of aberrant megakaryocytes (aMK). Transcriptionally and functionally,aMKPs mimic CG-AMKL cells and establish a self-renewal network with co-factors GATA2,ERG,and DLX3. In contrast,aMKs partially sustain regulators of MK maturation but fail to complete differentiation due to repression of factors like NFE2,SPI1,GATA1 and LYL1. These insights may inform new strategies for targeting AMKL cell states. Subject terms: Acute myeloid leukaemia,Cancer models View Publication