技术资料

To facilitate the characterization of unlabeled induced pluripotent stem cells (iPSCs) during culture and expansion,we developed an AI pipeline for nuclear segmentation and mitosis detection from phase contrast images of individual cells within iPSC colonies. The analysis uses a 2D convolutional neural network (U-Net) plus a 3D U-Net applied on time lapse images to detect and segment nuclei,mitotic events,and daughter nuclei to enable tracking of large numbers of individual cells over long times in culture. The analysis uses fluorescence data to train models for segmenting nuclei in phase contrast images. The use of classical image processing routines to segment fluorescent nuclei precludes the need for manual annotation. We optimize and evaluate the accuracy of automated annotation to assure the reliability of the training. The model is generalizable in that it performs well on different datasets with an average F1 score of 0.94,on cells at different densities,and on cells from different pluripotent cell lines. The method allows us to assess,in a non-invasive manner,rates of mitosis and cell division which serve as indicators of cell state and cell health. We assess these parameters in up to hundreds of thousands of cells in culture for more than 36 hours,at different locations in the colonies,and as a function of excitation light exposure. View Publication

BackgroundTimely resolution of innate immune responses activated by surgical intervention is crucial for patient recovery. While cytokines and innate immune cells are critical in inflammation resolution,the specific role of IL-18 in these processes remains controversial and underexplored.MethodsWe investigate determinants of successful recovery using peripheral blood samples from orthopedic surgery (ORT) patients (n?=?33) at T0 (before surgery),T1 (24 h after surgery) and T2 (3 days after surgery). Monocytes from ORT patients underwent immunophenotyping together with bulk transcriptomic analysis. We found that IL-18 strongly defines the recovery immune signature. These results were further validated in vitro by comparing IL-18 and TNF-? effects on monocytes,and in 3D human intestine organoids together with single cell (sc)-RNAseq analysis.ResultsTranscriptomics of ORT monocytes revealed upregulation of ITG family integrins,namely ITGB3 and ITGB5,CXCL family chemokines,notably CXCL1-3,CXCL5,and SCL/TAL1 factor controlling differentiation and migration,but not pro-inflammatory genes. Similar changes were observed in IL-18 stimulated healthy donor monocytes in vitro,including an increase in CD11b,CD64,and CD86 levels,accompanied by increased phosphorylation of Akt but not NF?B. These changes were attenuated in the presence of TNF-?,thus showing a unique role of IL-18 when acting alone without its most frequent paired cytokine TNF-?. We further confirmed that IL-18 induces monocyte-macrophage transition and migration using human intestinal organoids. Finally,TNF-?/IL-18 ratio showed a high predictive value of clinical severity in septic patients.ConclusionsWe propose a novel role of IL-18 on monocyte migration and macrophage transition characterizing successful orthopedic surgery recovery,as well as the ratio of IL-18/TNF-? as a novel marker of inflammation resolution,with potential implications for patient monitoring and therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06652-7. View Publication

BackgroundX-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation,which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides,animal models of XLRS have limitations in the study of XLRS. Here,we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS.MethodshiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently,we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy.ResultsROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor,retinoschisin (RS1) deficiency,and altered spontaneous activity compared with control ROs. Furthermore,the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.ConclusionsThe RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03767-4. 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

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

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

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

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

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

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

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

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