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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

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

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

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

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by loss-of-function mutations in the MECP2 gene,resulting in diverse cellular dysfunctions. Here,we investigated the role of the long noncoding RNA (lncRNA) NEAT1 in the context of MeCP2 deficiency using human neural cells and RTT patient samples. Through single-cell RNA sequencing and molecular analyses,we found that NEAT1 is markedly downregulated in MECP2 knockout (KO) cells at various stages of neural differentiation. NEAT1 downregulation correlated with aberrant activation of the mTOR pathway,abnormal protein metabolism,and dysregulated autophagy,contributing to the accumulation of protein aggregates and impaired mitochondrial function. Reactivation of NEAT1 in MECP2-KO cells rescued these phenotypes,indicating its critical role downstream of MECP2. Furthermore,direct RNA–RNA interaction was revealed as the key process for NEAT1 influence on autophagy genes,leading to altered subcellular localization of specific autophagy-related messenger RNAs and impaired biogenesis of autophagic complexes. Importantly,NEAT1 restoration rescued the morphological defects observed in MECP2-KO neurons,highlighting its crucial role in neuronal maturation. Overall,our findings elucidate lncRNA NEAT1 as a key mediator of MeCP2 function,regulating essential pathways involved in protein metabolism,autophagy,and neuronal morphology. View Publication

To investigate intestinal health and its potential disruptors in vitro,representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However,the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently,the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge,IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model,while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately,comparison to in vivo transcriptomics data showed limited similarities,which could be explained by essential differences in the study setups. Altogether,hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74802-w. View Publication

AbstractTGF-? signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-? family signaling is intrinsically executed remains not fully elucidated. Here,we show that HBO1,a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-? signaling in human embryonic stem cells (hESCs). HBO1?/? hESCs fail to response to TGF-? signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover,HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly,HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation,HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1,a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification. Graphical Abstract Graphical Abstract View Publication

Citron Kinase (CITK) is a protein encoded by the CIT gene,whose pathogenic variants underlie microcephalic phenotypes that characterize MCPH17 syndrome. In neural progenitors,CITK loss leads to microtubule instability,resulting in mitotic spindle positioning defects,cytokinesis failure,and accumulation of DNA double strand breaks (DSBs),ultimately resulting in TP53-dependent senescence and apoptosis. Although DNA damage accumulation has been associated with impaired homologous recombination (HR),the role of CITK in this process and whether microtubule dynamics are involved is still unknown. In this report we show that CITK is required for proper BRCA1 localization at sites of DNA DSBs. We found that CITK’s scaffolding,rather than its catalytic activity,is necessary for maintaining BRCA1 interphase levels in progenitor cells during neurodevelopment. CITK regulates the nuclear levels of HDAC6,a modulator of both microtubule stability and DNA damage repair. Targeting HDAC6 in CITK-deficient cells increases microtubule stability and recovers BRCA1 localization defects and DNA damage levels to that detected in controls. In addition,the CIT-HDAC6 axis is functionally relevant in a MCPH17 zebrafish model,as HDAC6 targeting recovers the head size phenotype produced by interfering with the CIT orthologue gene. These data provide novel insights into the functional interplay between HR and microtubule dynamics and into the pathogenesis of CITK based MCPH17,which may be relevant for development of therapeutic strategies. View Publication

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here,using human embryonic stem cell (hESC) lines,we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs),particularly pronounced in glia versus neurons. Employing a multiomics approach,we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally,RTT ASTs exhibited increased cytosolic amino acids under basal conditions,which were depleted upon increased energy demands. Notably,mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons,suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health. View Publication

PurposeOxaliplatin-induced peripheral neuropathy (OIPN) is a chronic,debilitating late effect following oxaliplatin treatment. Neurofilament light chain (NfL) is a structural protein found in nerve axons that was investigated upon oxaliplatin exposure in vitro and in vivo correlated to symptoms of OIPN in colorectal cancer patients receiving oxaliplatin.MethodsHuman sensory neurons,derived from induced pluripotent stem cells,were exposed to clinically relevant concentrations of oxaliplatin in vitro,with NfL concentrations measured in the cell medium. The prospective clinical study included patients with colorectal cancer undergoing chemotherapy therapy with or without oxaliplatin. Possible OIPN was defined as bilateral presence of numbness and/or presence of pricking sensations in the feet documented in an interview at the time of blood sampling prior to,3,and 6 months after initiating treatment.ResultsOxaliplatin exposure led to a dose-dependent NfL increase in vitro. In the clinical cohort of 30 patients (18 in the oxaliplatin group),NfL levels rose at 3 and 6 months compared to controls. NfL level changes correlated to OIPN symptoms at the 6-month timepoint (rho 0.81,p? View Publication