技术资料

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

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

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

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

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

Autism spectrum disorder (ASD) and congenital heart disease (CHD) frequently co-occur,yet the underlying molecular mechanisms of this comorbidity remain unknown. Given that children with CHD are identified as newborns,understanding which CHD variants are associated with autism could help select individuals for early intervention. Autism gene perturbations commonly dysregulate neural progenitor cell (NPC) biology,so we hypothesized that CHD genes disrupting neurogenesis are more likely to increase ASD risk. Therefore,we performed an in vitro pooled CRISPR interference screen to identify CHD genes disrupting NPC biology and identified 45 CHD genes. A cluster of ASD and CHD genes are enriched for ciliary biology,and perturbing any one of seven such genes (CEP290,CHD4,KMT2E,NSD1,OFD1,RFX3 and TAOK1) impairs primary cilia formation in vitro. In vivo investigation of TAOK1 in Xenopus tropicalis reveals a role in motile cilia formation and heart development,supporting its prediction as a CHD gene. Together,our findings highlight a set of CHD genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology. View Publication

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells,including functional ?-cells,have been based on a detailed understanding of the underlying developmental mechanisms. However,the final differentiation steps,leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells,remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells),which include a lack of ?-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP,INSeGFP/GCGmCHERRY) as well as ?-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive,insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions,we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and,using this approach,we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ?-cells without affecting the number of ?-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets. View Publication

Human GWAS have shown that obesogenic FTO polymorphisms correlate with lean mass,but the mechanisms have remained unclear. It is counterintuitive because lean mass is inversely correlated with obesity and metabolic diseases. Here,we use CRISPR to knock-in FTOrs9939609-A into hESC-derived tissue models,to elucidate potentially hidden roles of FTO during development. We find that among human tissues,FTOrs9939609-A most robustly affect human muscle progenitors’ proliferation,differentiation,senescence,thereby accelerating muscle developmental and metabolic aging. An edited FTOrs9939609-A allele over-stimulates insulin/IGF signaling via increased muscle-specific enhancer H3K27ac,FTO expression and m6A demethylation of H19 lncRNA and IGF2 mRNA,with excessive insulin/IGF signaling leading to insulin resistance upon replicative aging or exposure to high fat diet. This FTO-m6A-H19/IGF2 circuit may explain paradoxical GWAS findings linking FTOrs9939609-A to both leanness and obesity. Our results provide a proof-of-principle that CRISPR-hESC-tissue platforms can be harnessed to resolve puzzles in human metabolism. Human GWAS paradoxically linked FTO SNPs to both lean mass and sarcopenia/obesity. Here,Guang et al used CRISPR-edited stem cells to reveal that an obesogenic FTO SNP accelerates both muscle development and aging,by increasing RNA m6A demethylation. View Publication

Introduction: Africa,home to 1.4 billion people and the highest genetic diversity globally,harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However,African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections,limiting the exploration of population-specific disease mechanisms and therapeutic discoveries. Methods: To address this gap,we established an open-access African Somatic and Stem Cell Bank. Results: In this initial phase,we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency,genetic stability,differentiation potential,and Alzheimer's disease and Parkinson's disease risk variants. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-associated MAPT mutations (P301L and R406W). Discussion: This collection offers a renewable,genetically diverse resource to investigate disease pathogenicity in African populations,facilitating breakthroughs in neurodegenerative research,drug discovery,and regenerative medicine. Highlights: We established an open-access African Somatic and Stem Cell Bank. 10 induced pluripotent stem cell lines from five Nigerian ethnic groups were rigorously characterized. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-causing MAPT mutations. The African Somatic and Stem Cell Bank is a renewable,genetically diverse resource for neurodegenerative research. View Publication

BackgroundSpecific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases,including Parkinson’s disease (PD). However,the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here,according to the multiple-hit hypothesis,which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors,we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency,a genetic cause of PD,during lipopolysaccharide (LPS)-induced inflammation.MethodsUsing a combination of single-cell RNA-sequencing,bulk RNA-sequencing,multicolor flow cytometry and immunofluorescence analyses,we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives,we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs).ResultsBy excluding the contribution of other immune brain resident and peripheral cells,we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype,specially related to type II interferon and DNA damage response signaling,when compared with wildtype microglia,in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level,with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice,both at 6 and 24 h after acute inflammation,as also observed in BMDMs.ConclusionsTaken together,our results show that,under inflammatory conditions,PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses,which may play a causative role in PD onset and progression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03164-x. View Publication

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here,we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific,CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells,which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons,we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1?Mb,including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type,implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner. Scalable CRISPRa screening of cis-regulatory elements in non-cancer cell lines has proved challenging. Here,the authors describe a scalable,CRISPR activation screening framework to identify regulatory element-gene pairs in diverse cell types including cancer cells and neurons. View Publication

Neutrophils are critical for host defense against fungi. However,the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here,we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation,antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan,including an upregulation of the pentose phosphate pathway (PPP),similar to primary human neutrophils in vitro. Furthermore,we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively,these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections,and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation,limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus,demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively,these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity. View Publication