技术资料

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However,real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS),leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP),we created a novel,green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics,ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems,including human stem cell-derived neurons and cardiomyocytes,primary neurons and astrocytes,and mouse brain ex vivo and in vivo. These applications demonstrate oROS’s capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A?-putriscine-MAOB axis,highlighting the sensor’s relevance in validating neurodegenerative disease models. Lastly,we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool,offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology,with significant implications for understanding diseases associated with oxidative stress,such as cancer,neurodegenerative,and cardiovascular diseases. View Publication

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

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

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

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

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

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

Stem cell-derived ?-cells (SC-BCs) represent a potential source for curing diabetes. To date,in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation,indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here,we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ?-cells in vitro. To this aim,a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ?-cell markers,but did not improve ?-cell identity. In conclusion,the study describes the production of a PanMa hydrogel,which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs. View Publication