技术资料

The formation of mammalian synapses entails the precise alignment of presynaptic release sites with postsynaptic receptors but how nascent cell–cell contacts translate into assembly of presynaptic specializations remains unclear. Guided by pioneering work in invertebrates,we hypothesized that in mammalian synapses,liprin-? proteins directly link trans-synaptic initial contacts to downstream steps. Here we show that,in human neurons lacking all four liprin-? isoforms,nascent synaptic contacts are formed but recruitment of active zone components and accumulation of synaptic vesicles is blocked,resulting in ‘empty’ boutons and loss of synaptic transmission. Interactions with presynaptic cell adhesion molecules of either the LAR-RPTP family or neurexins via CASK are required to localize liprin-? to nascent synaptic sites. Liprin-? subsequently recruits presynaptic components via a direct interaction with ELKS proteins. Thus,assembly of human presynaptic terminals is governed by a hierarchical sequence of events in which the recruitment of liprin-? proteins by presynaptic cell adhesion molecules is a critical initial step. This paper identifies the evolutionarily conserved liprin-? protein family as key mediators of presynaptic assembly in human neurons. Their recruitment to sites formed by contacting neurons is the critical initial step that triggers presynaptic differentiation. View Publication

The emergence of the primitive streak,representing an organizing center for gastrulation,marks the mesendodermal lineage specification from epiblast,in which the epiblast cells undergo highly organized collective behaviors to form mesendodermal cells properly. Cell death is observed at the peri-gastrulation stage,especially in the primitive streak region. However,the dynamic and regulatory mechanism of cell death in the primitive streak formation is unclear. Here,we observed that a quick inhibition of the fast elevated cell death is coinciding with an accumulation of ?-catenin during the early stage of primitive streak induction from human embryonic stem cells (hESCs). Deficiency of ?-catenin in hESCs does not affect their self-renewal but cause robust cell death after primitive streak induction,while neuroectodermal differentiation remains unchanged. Overexpression of full-length ?-catenin in ?-catenin-deficient hESCs restores the cell death restriction during induction of primitive streak. Mechanistically,the ?-catenin-restricted cell death during primitive streak is transcription-independent. The accumulated ?-catenin traps casein kinase-1 in ?-catenin destruction complex following WNT activation via its ARM repeat domain,resulting in the inhibition of mTORC1 by stabilizing DEPTOR,subsequently attenuates mitochondrial translocation of p53 and enhances mitophagy to promote cell survival. Consistently,mTORC1 inhibition by rapamycin or RAD001 attenuates the cell death in ?-catenin-deficient cells during induction of primitive streak. In addition,only the ?-catenin retains activations of cell death restriction and transcriptional activity can promote hESCs to successfully differentiate into primitive streak and cardiomyocytes,suggesting that ?-catenin-restricted cell death safeguards the fate transition during the primitive streak induction via offering a crucial window for the accumulation of ?-catenin to induce lineage-specific genes. These findings provide new insights into the function and mechanisms by which ?-catenin coordinates the cell death and early lineage commitment. View Publication

In this study,Chen et al. describe two independent mechanisms that control ?-catenin levels in neuroglial cells and drive their proliferation. The work provides mechanistic insight into the impact of MEK activation resulting from the biallelic loss of NF1 or BRAF rearrangement in pediatric gliomas. The two major genomic alterations in pediatric pilocytic astrocytoma (PA) are NF1 loss and KIAA1549:BRAF rearrangement. Although these molecular changes result in increased MEK activity and tumor growth,it is not clear exactly how MEK controls human neuroglial cell proliferation. Leveraging human-induced pluripotent stem cells harboring these PA-associated alterations,we used a combination of genetic and pharmacological approaches to demonstrate that MEK-regulated cell growth is mediated by ?-catenin through independent mechanisms involving IRX2 control of CTNNB1 transcription and NPTX1 stabilization of ?-catenin protein levels. These results provide new mechanistic insights into MEK regulation of human brain cell function. View Publication

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases,regenerative medicine,and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs,which involves step-by-step differentiation of hiPSCs into definitive endoderm,anterior foregut endoderm,NKX2.1+ lung progenitors,and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs,expressing basal cell markers (NGFR,KRT5,and TP63),could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs,potentially applicable for gene therapy research. View Publication

Uncovering the stimulus-response histories that give rise to cell fates and behaviors is an area of great interest in developmental biology,tissue engineering,and regenerative medicine. A comprehensive accounting of cell experiences that lead to the development of organs and tissues can help us to understand developmental anomalies that may underly disease. Perhaps more provocatively,such a record can also reveal clues as to how to drive cell collective decision-making processes,which may yield predictable cell-based therapies or facilitate production of tissue substitutes for transplantation or in vitro screening of prospective therapies to mitigate disease. Toward this end,various methods have been applied to molecularly trace developmental trajectories and record interaction histories of cells. Typical methods involve artificial gene circuits based on recombinases that activate a suite of fluorescent reporters or CRISPR-Cas9 genome writing technologies whose nucleic acid-based record keeping serves to chronicle cell-cell interactions or past exposure to stimuli of interests. Exciting expansions of the synthetic biology toolkit with artificial receptors that permit establishment of defined input-to-output linkages of cell decision-making processes opens the door to not only record cell-cell interactions,but to also potentiate directed manipulation of the outcomes of such interactions via regulation of carefully selected transgenes. Here,we combine CRISPR-based strategies to genetically and epigenetically manipulate cells to express components of the synthetic Notch receptor platform,a widely used artificial cell signaling module. Our approach gives rise to the ability to conditionally record interactions between human cells,where the record of engagement depends on expression of a state-specific marker of a subset of cells in a population. Further,such signal-competent interactions can be used to direct differentiation of human embryonic stem cells toward pre-selected fates based on assigned synNotch outputs. We also implemented CRISPR-based manipulation of native gene expression profiles to bias outcomes of cell engagement histories in a targeted manner. Thus,we present a useful strategy that gives rise to both state-specific recording of cell-cell interactions as well as methods to intentionally influence products of such cell-cell exchanges. View Publication

BackgroundRecent studies highlight the critical role of microglia in neurodegenerative disorders,and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches,yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia.MethodsWe developed the hCSF1Bdes mouse line,which is suitable as a new transplantation model and available to be crossed to any disease model of interest. The hCSF1Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additionally,we crossed this model with two humanized App KI mice,the AppHu and the AppSAA. Flow cytometry,immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease.ResultsOur results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1Bdes mice without triggering a NK-driven immune response. Furthermore,we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1Bdes and the crosses with the Alzheimer’s disease knock-in model AppSAA and the humanized App knock-in control mice,AppHu are deposited with EMMA and fully accessible to the research community.ConclusionThe hCSF1Bdes mouse is available for both non-profit and for-profit organisations,facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00823-2. View Publication

SummaryIn Drosophila,long noncoding RNA Hsr? rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here,we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsr? via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally,we found that heat shock stress promoted cell differentiation,which is conserved in mammalian cells through paraspeckles,resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall,our findings confirm the role of Hsr? and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies. Graphical abstract Highlights•LncRNA Hsr? is differentially expressed in different cell types of fly midguts•Omega speckles nucleate TPBH and promote the differentiation of ISCs to ECs•Heat shock treatment induces the assembly of omega speckles and paraspeckles•Heat shock treatment accelerates the differentiation of fly midguts and human iPSCs Molecular biology; Cell biology; Developmental biology View Publication

Neural stem cells have intact innate immune responses that protect them from virus infection and cell death. Yet,viruses can antagonize such responses to establish neuropathogenesis. Using a forebrain organoid model system at two developmental time points,we identified that neural stem cells,in particular radial glia,are basally primed to respond to virus infection by upregulating several antiviral interferon-stimulated genes. Infection of these organoids with a neuropathogenic Enterovirus-D68 strain,demonstrated the ability of this virus to impede immune activation by blocking interferon responses. Together,our data highlight immune gene signatures present in different types of neural stem cells and differential viral capacity to block neural-specific immune induction.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03275-5. View Publication

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here,we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency,encompassing an eight-day time course with modulation of WNT,BMP,and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm,neural crest,and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas,evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together,this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology. Shen et al. report a method for multiplexing isogenic iPSCs for single-cell RNA-seq. With it,they created an atlas of in vitro differentiation and identified TMEM88 as a regulator of cardiovascular development,impacting blood pressure in adult mice. View Publication

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity. Calcium is an important signal transducer that regulates key cellular processes,but its role in regulating CM proliferation is incompletely understood. Here we show a robust pathway for new calcium signaling-based cardiac regenerative strategies. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC) induced the CM cell cycle. Furthermore,overexpression of Ras-related associated with Diabetes (RRAD),an endogenous inhibitor of LTCC,induced CM cell cycle activity in vitro,in human cardiac slices,and in vivo. Mechanistically,LTCC inhibition by RRAD or nifedipine induced CM cell cycle by modulating calcineurin activity. Moreover,ectopic expression of RRAD/CDK4/CCND in combination induced CM proliferation in vitro and in vivo,improved cardiac function and reduced scar size post-myocardial infarction. View Publication

Human induced pluripotent stem cells (iPSCs) have great potential in research,but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here,we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD,NANOG,SPP1),endoderm (CER1,EOMES,GATA6),mesoderm (APLNR,HAND1,HOXB7),and ectoderm (HES5,PAMR1,PAX6). Using these genes,we develop a machine learning-based scoring system,“hiPSCore”,trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time,subjectivity,and resource use,thus enhancing iPSC quality for scientific and medical applications. Quality control,including pluripotency testing of human iPSCs lacks standardization. Here,authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality. View Publication

Under specific conditions,cultured human embryonic stem cells (hESCs) corresponding to primed post-implantation epiblasts can be converted back to a ‘naïve pluripotency’ state that resembles the pre-implantation epiblasts. The core pluripotency factor OCT4 is known to be crucial in regulating different states of pluripotency,but its potential regulatory role in human naïve pluripotency remains unexplored. In this study,we systematically mapped out phosphorylation sites in OCT4 protein that are differentially phosphorylated between two states of pluripotency,and further identified ATR as a key kinase that phosphorylated OCT4 in naïve but not primed hESCs. The kinase activity levels of ATR in naïve hESCs were higher than those in primed hESCs. Ablating cellular ATR activity significantly halted the induction of naïve hESCs from their primed counterparts,and increased early apoptotic death of naïve hESCs upon UV and CPT treatment. Thus,our work reveals the importance of ATR activity in safeguarding human naïve pluripotency,and implicates a potential association of OCT4 phosphorylation,DNA damage sensing and repairing system in regulating different states of pluripotency during early development. View Publication