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During a heart attack,ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment,thereby shifting focus to cell-free therapies. Consequently,we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature,efficacy in previous studies,and easy obtainability from minimally invasive techniques. Nonetheless,using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome,regardless of donor health,would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors,passaged 6–10 times,underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content,independent of donor health status,had elevated expression of proteins involved in cell survival pathways. In IR conditions,iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05),cell viability (p < 0.001),and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently,only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore,secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences. View Publication

BackgroundThe expansion of GGC repeats (typically exceeding 60 repeats) in the 5’ untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs),such as neuronal intranuclear inclusion disease (NIID),frontotemporal dementia (FTD),essential tremor (ET),and Parkinson’s disease (PD). These disorders share common clinical manifestations,including parkinsonism,dementia,seizures,and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats,particularly those with AGC-encoded serine insertions,have been reported to be associated with PD; however,the functional implications of these intermediate repeats with serine insertion remain unexplored.MethodsHere,we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs.ResultsOur findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice,32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics,including the formation of ?-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally,32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways,both of which are critical for mitochondrial function. Notably,genes associated with myelin sheath components,including MBP and MOG,were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex.ConclusionsOur in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C,leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00780-2. View Publication

This paper presents a 360°,size-adjustable microelectrode array (MEA) system for the long-term electrophysiological monitoring of cerebral organoids derived from human pluripotent stem cells. The system consists of eight independently positionable multielectrode probes,each carrying eight electrodes arranged vertically. This configuration resulted in 64 recording channels surrounding the organoid. The multielectrode probes were mounted on custom-designed miniature manipulators with three degrees of freedom. This setup enabled positioning and contact with organoids of varying sizes (approximately 1–3.7 mm in diameter). The design allowed circumferential access and facilitated standard incubator-based cultivation without disrupting the recording setup. Fabricated using flexible printed circuit technology,this MEA system offers relatively low production costs. It is also amenable to widespread implementation in laboratory settings. Experimental results demonstrated the successful recording of neuronal activity,including spike detection and signal stability,over 2 weeks of continuous organoid culture. These results suggests that the three-dimensional system provides broad spatial coverage and supports long-term monitoring for basic biomedical research. It also holds potential for future applications such as biohybrid computing. View Publication

Genetic variants often produce complex phenotypic effects that confound current assays and predictive models. We developed Variant in situ sequencing (VIS-seq),a pooled,image-based method that measures variant effects on molecular and cellular phenotypes in diverse cell types. Applying VIS-seq to ~3,000 LMNA and PTEN variants yielded high-dimensional morphological profiles that captured variant-driven changes in protein abundance,localization,activity and cell architecture. We identified gain-of-function LMNA variants that reshape the nucleus and autism-associated PTEN variants that mislocalize. Morphological profiles predicted variant pathogenicity with near-perfect accuracy and distinguished autism-linked from tumor syndrome-linked PTEN variants. Most variants impacted a multidimensional continuum of phenotypes not recapitulated by any single functional readout. By linking protein variation to cell images at scale,we illuminate how variant effects cascade from molecular to subcellular to cell morphological phenotypes,providing a framework for resolving the complexity of variant function. View Publication

Microglia are the immune cells in the central nervous system (CNS) and become pro-inflammatory/activated in Alzheimer’s disease (AD). Cell surface glycosylation plays an important role in immune cells; however,the N-glycosylation and glycosphingolipid (GSL) signatures of activated microglia are poorly understood. Here,we study comprehensively combined transcriptomic and glycomic profiles using human induced pluripotent stem cells-derived microglia (hiMG). Distinct changes in N-glycosylation patterns in amyloid-? oligomer (A?O) and LPS-treated hiMG were observed. In A?O-treated cells,the relative abundance of bisecting N-acetylglucosamine (GlcNAc) N-glycans decreased,corresponding with a downregulation of MGAT3. The sialylation of N-glycans increased in response to A?O,accompanied by an upregulation of genes involved in N-glycan sialylation (ST3GAL4 and 6). Unlike A?O-induced hiMG,LPS-induced hiMG exhibited a decreased abundance of complex-type N-glycans,aligned with downregulation of mannosidase genes (MAN1A1,MAN2A2,and MAN1C1) and upregulation of ER degradation related-mannosidases (EDEM1-3). Fucosylation increased in LPS-induced hiMG,aligned with upregulated fucosyltransferase 4 (FUT4) and downregulated alpha-L-fucosidase 1 (FUCA1) gene expression,while sialofucosylation decreased,aligned with upregulated neuraminidase 4 (NEU4). Inhibition of sialylation and fucosylation in A?O- and LPS-induced hiMG alleviated pro-inflammatory responses. However,the GSL profile did not exhibit significant changes in response to A?O or LPS activation,at least in the 24-hour stimulation timeframe. A?O- and LPS- specific glycosylation changes could contribute to impaired microglia function,highlighting glycosylation pathways as potential therapeutic targets for AD.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-96596-1. View Publication

Oropouche fever is a re-emerging global viral threat caused by infection with Oropouche orthobunyavirus (OROV). While disease is generally self-limiting,historical and recent reports of neurologic involvement highlight the importance of understanding the neuropathogenesis of OROV. In this study,we characterize viral replication kinetics in neurons and microglia derived from immortalized,primary,and induced pluripotent stem cell-derived cells,which are all permissive to infection. We demonstrate that ex vivo rat brain slice cultures can be infected by OROV and produce antiviral cytokines and chemokines,including IL-6,TNF-? and IFN-?,which introduces an additional model to study viral kinetics in the central nervous system. These findings provide additional insight into OROV neuropathogenesis and in vitro modeling strategies for a newly re-emerging arbovirus. View Publication

Mutations in the microtubule binding motor protein,kinesin family member 5A (KIF5A),cause the fatal motor neuron disease,Amyotrophic Lateral Sclerosis. While KIF5 family members transport a variety of cargos along axons,it is still unclear which cargos are affected by KIF5A mutations. We generated KIF5A null mutant human motor neurons to investigate the impact of KIF5A loss on the transport of various cargoes and its effect on motor neuron function at two different timepoints in vitro. The absence of KIF5A resulted in reduced neurite complexity in young motor neurons (DIV14) and significant defects in axonal regeneration capacity at all developmental stages. KIF5A loss did not affect neurofilament transport but resulted in decreased mitochondria motility and anterograde speed at DIV42. More prominently,KIF5A depletion strongly reduced anterograde transport of SFPQ-associated RNA granules in DIV42 motor neuron axons. We conclude that KIF5A most prominently functions in human motor neurons to promote axonal regrowth after injury as well as to anterogradely transport mitochondria and,to a larger extent,SFPQ-associated RNA granules in a time-dependent manner. View Publication

Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate,identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here,we present DELVE,an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories. In contrast to previous work,DELVE uses a bottom-up approach to mitigate the effects of confounding sources of variation,and instead models cell states from dynamic gene or protein modules based on core regulatory complexes. Using simulations,single-cell RNA sequencing,and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation,we demonstrate how DELVE selects features that better define cell-types and cell-type transitions. DELVE is available as an open-source python package: https://github.com/jranek/delve. Characteristic genes or proteins driving continuous biological processes are difficult to uncover from noisy single-cell data. Here,authors present DELVE,an unsupervised feature selection method to identify core molecular features driving cell fate decisions. View Publication

Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7,a recently developed cytosine deaminase,exhibits high activity across a broad protospacer range but induces unintended off-target effects,including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here,we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this,we engineer two Sdd7 variants,Sdd7e1 and Sdd7e2,enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing,narrowed editing windows,and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity,nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2,especially when delivered via eVLP,as high-fidelity CBEs poised for safe,precise therapeutic genome editing. CRISPR base editors enable precise DNA changes but often cause off-target edits. Here,authors engineer two Sdd7 variants that minimize bystander and off-target mutations and show enhanced precision when delivered as ribonucleoproteins via engineered virus-like particles. View Publication

SummaryThe cornea and sclera are distinct adjacent tissues,yet their stromal cells originate from common neural crest cells (NCCs). Sclerocornea is a disease characterized by an indistinguishable boundary between the cornea and sclera. Previously,we identified a RAD21 mutation in a sclerocornea pedigree. Here,we investigated the impacts of RAD21 on NCC activities during eye development. RAD21 deficiency caused upregulation of PCDHGC3. Both RAD21 knockdown and PCDHGC3 upregulation disrupted the migration of NCCs. Transcriptome analysis indicated that WNT9B had 190.9-fold higher expression in scleral stroma than in corneal stroma. WNT9B was also significantly upregulated by both RAD21 knockdown and PCDHGC3 overexpression,and knock down of WNT9B rescued the differentiation and migration of NCCs with RAD21 deficiency. Consistently,overexpressing wnt9b in Xenopus tropicalis led to ocular developmental abnormalities. In summary,WNT9B is a determinant factor during NCC differentiation into corneal keratocytes or scleral stromal cells and is affected by RAD21 expression. Graphical abstract Highlights•Established a stable differentiation protocol from hESCs to corneal keratocytes•RAD21 deficiency affected the proliferation and migration ability of NCCs•Increased scleral markers after RAD21 knockdown during NCC differentiation to cornea•WNT9B is a crucial mediator during ocular NCC differentiation Cell biology; Developmental biology View Publication

BackgroundAtrial fibrillation has an estimated prevalence of 1.5–2%,making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic,as well as local,inflammatory processes has been reported. However,the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology,a direct,causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes,to propose a mechanistic link between inflammation and atrial fibrillation.MethodsTwo hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic,pacing-free,atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity,electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes.ResultsMulti electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A,KCNA5,ATP1A1,and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes,including reduced beat irregularity,improved conduction,and reversed RNA expression profiles.ConclusionsThis study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia,documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further,beat rate irregularity could be alleviated using glucocorticoids. Overall,these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03814-0. View Publication

Background & aimsHumans with WNT2B deficiency have severe intestinal disease,including significant inflammatory injury,highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.MethodsWe investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon,and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue.ResultsMice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased,epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6,and biopsy tissue from humans showed increased neutrophils.ConclusionsWNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation,particularly in the colon. Graphical abstract View Publication