技术资料

Breast cancer is the most frequently diagnosed cancer worldwide,constituting 15% of cases in 2023. The predominant cause of breast cancer-related mortality is metastasis,and a lack of metastasis-targeted therapies perpetuates dismal outcomes for late-stage patients. By using meiotic genetics to study inherited transcriptional network regulation,we have identified,to the best of our knowledge,a new class of “essential expression-restricted” genes as potential candidates for metastasis-targeted therapeutics. Building upon previous work implicating the CCR4-NOT RNA deadenylase complex in metastasis,we demonstrate that RNA-binding proteins NANOS1,PUM2,and CPSF4 also regulate metastatic potential. Using various models and clinical data,we pinpoint Smarcd1 mRNA as a target of all three RNA-BPs. Strikingly,both high and low expression of Smarcd1 correlate with positive clinical outcomes,while intermediate expression significantly reduces the probability of survival. Applying the theory of “essential genes” from evolution,we identify 50 additional genes that require precise expression levels for metastasis to occur. Specifically,small perturbations in Smarcd1 expression significantly reduce metastasis in mouse models and alter splicing programs relevant to the ER+/HER2-enriched breast cancer. Identification subtype-specific essential expression-restricted metastasis modifiers introduces a novel class of genes that,when therapeutically “nudged” in either direction,may significantly improve late-stage breast cancer patients. Subject terms: Metastasis,Cancer genetics,Breast cancer View Publication

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However,lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation remains poorly understood,and access to well-characterized placental cells for biomedical research is limited,largely depending on fetal tissues or cancer cell lines. Here,we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated syncytiotrophoblast (STB),followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological,biochemical,genomics,epigenomics,and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development,infertility,and pregnancy-associated diseases. Subject areas: Natural sciences,Biological sciences,Cell biology,Stem cells research View Publication

Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades,a notable portion of children still confronts challenges such as treatment resistance and recurrence,leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research,we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. Employing short hairpin RNA (shRNA) techniques,we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq,CUT&Tag,and immunoprecipitation assays,we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG,ETV6,IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL,with MYB identified as a significant downstream target of LDB1. To sum up,our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG,ETV6,and IRF1 to modulate the expression of downstream effector genes. Furthermore,LDB1 controls MYB through remote enhancer modulation,providing valuable mechanistic insights into its involvement in the progression of T-ALL. The online version contains supplementary material available at 10.1186/s13046-024-03199-1. View Publication

CRISPR-based genome editing of pseudogene-associated disorders,such as p47 phox -deficient chronic granulomatous disease (p47 CGD),is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes,NCF1B and NCF1C,in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering,a droplet digital PCR-based method identified cells with altered copy numbers,spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context,such as the presence of homologous regions,whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing. Subject terms: CRISPR-Cas9 genome editing,Targeted gene repair,Haematopoietic stem cells View Publication

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types,including pancreatic insulin-producing β cells,which are crucial for developing therapies for diabetes. However,current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here,we report that highly selective tankyrase inhibitors,such as WIKI4,significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development,but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications. Subject terms: Stem-cell differentiation,Organogenesis,Type 1 diabetes View Publication

Addiction is known to occur through the consumption of substances such as pharmaceuticals,illicit drugs,food,alcohol and tobacco. These addictions can be viewed as drug addiction,resulting from the ingestion of chemical substances contained in them. Multiple neural networks,including the reward system,anti‐reward/stress system and central immune system in the brain,are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted,methods for assessing addiction have not been established. In this study,we aimed to develop an in vitro method for assessing the addiction of compounds,as an alternative to animal experiments,using human iPS cell‐derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non‐addictive compounds,demonstrating the ability to estimate addiction of compound. Additionally,conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine,dopamine and GABA) due to chronic administration of addictive compounds,suggesting the potential interpretation of these expression changes as addiction‐like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell‐derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks. View Publication

Extracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising,natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture,human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However,hiPSC-EVs only have a moderate ability for brain delivery. Herein,we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor. An RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging. Here we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype,express pluripotency markers,and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways,compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency. Taken together,we have established stable,genetically engineered hiPSCs for producing EVs with RVG expression,offering the improved ability for brain-targeted drug delivery. The online version contains supplementary material available at 10.1186/s13287-024-03955-2. View Publication

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here,we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data,FN mutations disrupted protein secretion from MSCs,causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP,HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol,and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation,reduced chondrogenic markers,and compromised cell proliferation in mutant cells. Moreover,FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression,crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. The online version contains supplementary material available at 10.1007/s00018-024-05444-4. View Publication

In calcium imaging studies,Ca 2+ transients are commonly interpreted as neuronal action potentials (APs). However,our findings demonstrate that robust optical Ca 2+ transients primarily stem from complex “AP-Plateaus”,while simple APs lacking underlying depolarization envelopes produce much weaker photonic signatures. Under challenging in vivo conditions,these “AP-Plateaus” are likely to surpass noise levels,thus dominating the Ca 2+ recordings. In spontaneously active neuronal culture,optical Ca 2+ transients (OGB1-AM,GCaMP6f) exhibited approximately tenfold greater amplitude and twofold longer half-width compared to optical voltage transients (ArcLightD). The amplitude of the ArcLightD signal exhibited a strong correlation with the duration of the underlying membrane depolarization,and a weaker correlation with the presence of a fast sodium AP. Specifically,ArcLightD exhibited robust responsiveness to the slow “foot” but not the fast “trunk” of the neuronal AP. Particularly potent stimulators of optical signals in both Ca 2+ and voltage imaging modalities were APs combined with plateau potentials (AP-Plateaus),resembling dendritic Ca 2+ spikes or “UP states” in pyramidal neurons. Interestingly,even the spikeless plateaus (amplitude > 10 mV,duration > 200 ms) could generate conspicuous Ca 2+ optical signals in neurons. Therefore,in certain circumstances,Ca 2+ transients should not be interpreted solely as indicators of neuronal AP firing. Subject terms: Biological techniques,Biophysics,Neuroscience,Physiology View Publication

Poor graft function (PGF),characterized by myelosuppression,represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However,the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF,as well as after infected by HCMV AD 169 strain in vitro,characterized by decreased cell proliferation,tube formation,migration and hematopoietic support,and increased apoptosis and secretion of TGF-β1. We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover,HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-β1 secretion by BM-EPCs. HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-β1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism,ultimately leading to compromised support for hematopoietic progenitors by BM EPCs,which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF. View Publication

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance,they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors,which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT),a well-known checkpoint receptor involved in T-cell exhaustion,has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here,we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability,are rapidly and efficiently taken up by cells,and can be easily incorporated into cell product manufacturing. INTASYL PH-804,which targets TIGIT,suppresses TIGIT mRNA and protein expression in NK cells,resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition,thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy,particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x. View Publication

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2 ) drives myeloid malignancy initiation and progression 1 – 3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4,5 . However,the open chromatin observed in TET2-deficient mouse embryonic stem cells,leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6,which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub,more open chromatin and increased transcription in stem cells. TET2- mutant human leukaemia becomes dependent on this gene activation pathway,with MBD6 depletion selectively blocking proliferation of TET2 -mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together,our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies. Subject terms: Molecular biology,Chromatin View Publication