技术资料

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

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context,the roles of microRNAs have received less attention. In the present study,we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients,showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1),which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia. Synopsis The involvement of microRNAs in the pathogenesis of schizophrenia is not well-understood. This study shows that miR-99b-5p regulates Z-DNA binding protein 1 (Zbp1) to control inflammatory responses in microglia and the development of schizophrenia-like symptoms in mice. miR-99b-5p is downregulated in the blood and brains of schizophrenia patients.miR-99b-5p inhibition induces schizophrenia-like phenotypes in mice and microglial inflammation.Zbp1 is a novel miR-99b-5p target in microglia.Zbp1 antisense oligos ameliorate the pathological outcomes of decreased miR-99b-5p levels. Dysregulation of a novel miR-99b-5p-Zbp1 (Z-DNA binding protein 1) pathway in microglia induces inflammatory responses and schizophrenia-like phenotypes in mice. View Publication

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless,a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process,including hypoxic stress,fibrosis,and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions,leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology,demonstrating that the phased combination of TGF? inhibitor SB431542,Rho kinase inhibitor Y27632,and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction,providing a crucial platform for discovering therapeutic targets for ischemic heart disease. Engineered human models of hearts are needed to study pathology and repair. Here,the authors develop a model which replicates the phased remodelling process. The model is then used to study signalling pathway modulators for their therapeutic potential in a mini-repair model. View Publication

Background: Takotsubo cardiomyopathy (TTC) is marked by an acute,transient,and reversible left ventricular systolic dysfunction triggered by stress,with endothelial dysfunction being one of its pathophysiological mechanisms. However,the precise molecular mechanism underlying the interaction between endothelial cells and cardiomyocytes during TTC remains unclear. This study reveals that exosomal miRNAs derived from endothelial cells exposed to catecholamine contribute to ion channel dysfunction in the setting of TTC. Methods: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with epinephrine (Epi) or exosomes (Exo) from Epi-treated human cardiac microvascular endothelial cells (HCMECs) or Exo derived from HCMECs transfected with miR-126-3p. The immunofluorescence staining,flow cytometry,qPCR,single-cell contraction,intracellular calcium transients,patch-clamp,dual luciferase reporter assay and western blot were performed for the study. Results: Modeling TTC with high doses of epinephrine (Epi) treatment in hiPSC-CMs shows suppression of depolarization velocity (Vmax),prolongation of action potential duration (APD),and induction of arrhythmic events. Exo derived from HCMECs treated with Epi (Epi-exo) mimicked or enhanced the effects of Epi. Epi exposure led to elevated levels of miR-126-3p in both HCMECs and their exosomes. Exo enriched with miR-126-3p demonstrated similar effects as Epi-exo,establishing the crucial role of miR-126-3p in the mechanism of Epi-exo. Dual luciferase reporter assay coupled with gene mutation techniques identified that miR-126-3p was found to target the regulator of G-protein signaling 3 (RGS3) gene. Western blot and qPCR analyses confirmed that miR-126-3p-mimic reduced RGS3 expression in both HCMECs and hiPSC-CMs,indicating miR-126-3p inhibits RGS3 signaling. Additionally,miR-126-3p levels were significantly higher in the serum of TTC patients compared to healthy controls and patients who had recovered from TTC. Conclusions: Our study is the first to reveal that exosomal miR-126-3p,originating from endothelial cells,contributes to ion channel dysfunction by regulating RGS3 signaling in cardiomyocytes. These findings provide new perspectives on the pathogenesis of TTC and suggest potential therapeutic targets for treatment. 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

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues,including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However,despite the critical roles of PAX1 in embryonic development and diseases,detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays,qRT-PCR and in situ hybridization in HEK293FT,HCT116 cells and zebrafish embryos,respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation,flow cytometry,high-throughput sequencing data analysis,and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere,we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically,PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2,thus perturbing TCF7L2 SUMOylation level,further reducing its transcriptional activity and protein stability. Moreover,we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells,which give rise to the thymus epithelium,by inhibiting Wnt signaling. Importantly,our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation,thus providing insights for the molecular basis of PAX1 associated SCID,offering better understanding of the behavior of PAX1 in embryogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01629-3. 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

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here,we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules,retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR),resulted in rapid,synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model,and they possess all the features necessary to be considered valid. Collectively,our data demonstrate a facile,scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta. View Publication