技术资料

SummaryThe ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus,the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay,we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons,a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival. Graphical abstract Highlights•rs7132908 is a causal variant at the chr12q13 obesity locus•rs7132908 regulates nearby effector genes with allele and cell-type specificity•Obesity risk allele decreases generation of neurons that regulate appetite A locus on chr12q13 is strongly associated with childhood obesity by genome-wide associate studies. Littleton et al. identified a causal variant at this locus,which regulates gene expression in neural cell types. The obesity risk allele also decreased the proportion of appetite-regulating hypothalamic neurons generated by stem cell differentiation. View Publication

For genome editing,the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins,which enable the targeted manipulation of transcriptomes and epigenomes,remain significantly less accessible. Here,we describe the production,delivery,and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency,even for genes that are strongly silenced,such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies,gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing,enabling undiminished gene activation of multiple genes simultaneously. Applying these insights,we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally,we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors. View Publication

Human development relies on the correct replication,maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages,and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells,we identify that several patterning signals—including WNT,BMP,and FGF—converge into the modulation of DNA replication stress and damage during S-phase,which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing,DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers,but re-emerges in neural progenitors. In particular,we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis,which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development. Here the authors show that the patterning signals WNT,BMP,and FGF control chromosome segregation fidelity during early lineage specification and neurogenesis,which could provide a rationale for the spatio-temporal distribution of genomic mosaicism during human development. View Publication

SummaryCardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage,which must be repaired by DNA damage response proteins. However,the impact of DNA damage on global cardiomyocyte protein abundance,and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control,and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing,chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD,such as arrhythmia are enriched in specific DOX-correlated modules,DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead,they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD,and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation. View Publication

Neurological damage caused by peripheral nerve injury can be devastating and is a common neurological disorder that,along with muscle disorders,reduces the quality of life. Neural crest cells (NCCs) are a transient cell population that occurs during embryogenesis,can differentiate into various cells upon transplantation,and has potential therapeutic effects on neurological diseases. However,there are limitations to cell therapy,such as uncontrolled differentiation and tumor formation. Extracellular vesicles (EVs),which are non-cellular potential therapeutic candidates,are nanosized membrane-bound vesicles. Studies have been reported using starch cells derived from neural cells,such as neural stem cells,because they are involved in cell-to-cell communication and carry a variety of bioactive molecules. We investigated the effects of EVs isolated from NCCs on neuronal cell death and inflammation. Additionally,we overexpressed the nerve growth factor (NGF),which is involved in neural cell growth and proliferation,in NCCs to further investigate the effects of EVs containing NGF. NCCoe-NGF-EVs showed neuroprotective and regenerative properties by modulating inflammatory pathway,promoting Schwann cell activation,and enhancing remyelination. In vitro studies on NCCoe-NGF-EVs suppressed pro-inflammatory cytokines and reduced oxidative stress-induced neuronal apoptosis through NF-?B pathway inhibition and ERK,AKT signal activation. We also evaluated the effect of EVs on neuropathy by performing in vivo study. Our results suggest that NCCoe-NGF-EV had neuroprotective effects by reducing neuronal apoptosis and promoting neuronal proliferation based on neurite outgrowth and anti-inflammation effects treated with NCCoe-NGF-EVs. In addition,NCCoe-NGF-EVs were protected muscle loss caused by peripheral nerve injury. NCCoe-NGF-EV induced regeneration of damaged nerves and inhibited cell death through anti-inflammatory effects. This study suggests the potential of NGF-enriched EVs as non-cellular therapeutic platform for peripheral neuropathies and other neuroinflammatory disorders. View Publication

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle,which are among the most important livestock species for milk and meat production. Here,we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First,we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4),followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist,bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes,including PRDM1/BLIMP1,TFAP2C,SOX17,and NANOS3,were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle. View Publication

Venoms comprise highly sophisticated bioactive molecules modulating ion channels,receptors,coagulation factors,and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research,we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis,pore formation,and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements,we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic,ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms,we found that the pore-forming latrotoxin ?-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes,while the lytic bee peptide melittin is not selective. Furthermore,evaluation of snake venoms showed that Elapid species induced rapid membrane lysis,while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits,aligning with clinical observations at venom level,beyond discriminating among ion pore-forming,membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic,cardiotoxic and neurotoxic effects,and assist in identifying venom components that hold the potential to benefit humankind. Graphical abstractImage 1 Highlights•Optimization of bioassays to study venoms strengthens the discovery of novel drugs and envenomation treatments•We developed a high-content bioassay measuring DNA and [Ca2+]i that investigates multiple mechanisms in venom biology•This bioassay monitored membrane integrity,ion channels and ion pore formation to unravel venom's mechanism of action•We found the latrotoxin ?-Lt1a strikingly prefers neuron-like cells while the ?-helical melittin is non-selective•Evaluation of Elapid and Viper snake venoms demonstrates that this bioassay predicts the phylogeny and clinical findings View Publication

Liver disease secondary to an inborn or genetic error of metabolism is a rare group of conditions often associated with chronic ill health and reduced survival. Curative treatment is mainly limited to liver transplantation with major long-term risks. Cell therapy is a promising alternative,but current approaches are ineffective. To develop histotripsy,a non-invasive high-intensity ultrasound procedure for liver tissue mechanical ablation,combined with hepatocyte stem cell implantation as a novel method of reversing liver failure from genetic disease. This study assessed the safety and feasibility of this approach in healthy rodents. Under general anaesthesia,adult rats (n = 12) underwent laparotomy and ultrasound histotripsy to the exposed liver. Around 1 million cells were injected into a single histotripsy cavity in each animal under direct vision (n = 10) with two receiving only histotripsy without cell injection. On completion of cell implant,haemostasis was secured,laparotomy incision closed and the animals recovered. Groups of animals were terminated immediately and after 4 h,8 h,24 h,4 days and 7 days. Liver and vital organs were assessed for procedure-related injuries and evidence of viable implanted cells by histology and immunohistochemistry. All animals successfully recovered,and no complication was observed throughout the study. Created cavities were successfully identified in histological analysis of rat. The presence of human cells was verified using anti-human nuclei antibody confirming successful implantation of liver organoids into decellularised cavities. In this feasibility study,we demonstrated suitability of histotripsy to create decellularised cavities in liver parenchyma. In addition,feasibility of direct transplantation of undissociated liver organoids into the created cavities was demonstrated as a potential approach to treat inborn liver disease by creating nodules of healthy cells capable of performing loss metabolic function. Therapeutic efficacy of this approach will be evaluated in an upcoming study. Graphical Abstract View Publication

N-terminal acetyltransferases including NAA10 catalyze N-terminal acetylation,an evolutionarily conserved co- and post-translational modification. However,little is known about the role of N-terminal acetylation in cardiac homeostasis. To gain insight into cardiac-dependent NAA10 function,we studied a previously unidentified NAA10 variant p.(Arg4Ser) segregating with QT-prolongation,cardiomyopathy,and developmental delay in a large kindred. Here,we show that the NAA10R4S variant reduced enzymatic activity,decreased NAA10-NAA15 complex formation,and destabilized the enzymatic complex N-terminal acetyltransferase A. In NAA10R4S/Y-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs),dysregulation of the late sodium and slow delayed rectifier potassium currents caused severe repolarization abnormalities,consistent with clinical QT prolongation. Engineered heart tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased contractile force and sarcomeric disorganization,consistent with the pedigree’s cardiomyopathic phenotype. Proteomic studies revealed dysregulation of metabolic pathways and cardiac structural proteins. We identified small molecule and genetic therapies that normalized the phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines the roles of N-terminal acetylation in cardiac regulation and delineates mechanisms underlying QT prolongation,arrhythmia,and cardiomyopathy caused by NAA10 dysfunction. N-terminal acetylation dysregulation in the heart causes severe arrhythmia and cardiomyopathy. The authors show that stem cell models demonstrate ion channel trafficking defects and sarcomeric disarray as the underlying mechanisms,with gene therapy reversing both phenotypes View Publication

ABSTRACTThe gut microbiota and the immune system are closely connected,influencing early?life brain development. Brain?derived neurotrophic factor (BDNF),crucial for neuronal development,has been demonstrated to be produced by certain immune cells. However,the modulation of BDNF during bacterial antigen and metabolite challenge remains elusive. We investigate the effects of bacterial?derived antigens and metabolites on BDNF secretion in human PBMCs. Although BDNF levels were altered during stimulation,a specific cellular origin of BDNF within PBMCs was indeterminate. Positive magnetic separation of monocytes eliminated both the stimulant?induced BDNF secretion and reduced monocyte?platelet aggregates. Conversely,elevated platelet counts significantly increased BDNF levels,indicating that platelets,when interacting with monocytes and exposed to bacterial antigens,are likely the dominant source of BDNF in PBMC cultures. As previously described,platelets are a crucial source of circulating peripheral blood BDNF. Our findings emphasize the importance of the interplay between immune?blood cell complexes during microbial stimulation in regulating BDNF levels. This highlights the necessity of investigating such interactions to better understand the early?life gut?brain axis. Bacterial antigens primarily induce BDNF release from platelets interacting with monocytes in PBMCs. This interplay underscores how immune?blood cell complexes shape BDNF levels which may impact early human development. View Publication

The oral mucosa plays an important role in maintaining oral and systemic health by protecting the body from harmful environmental stimuli and pathogens. Current reconstructed human gingiva models (RhG) serve as valuable testing platforms for safety and efficacy testing of dental materials,however they lack important phenotypic characteristics typical of the gingival epithelium. We aimed to determine whether incorporating induced pluripotent stem cells (iPSCs) into the hydrogel of a cell-line RhG (reconstructed epithelium on fibroblast-populated-hydrogel) would improve its phenotype. Immortalized human gingival fibroblasts were resuspended with and without iPSCs in collagen-fibrin hydrogels and gingival keratinocytes were seeded on top of the hydrogels to construct RhGs. RhGs were cultured at air-liquid interface for 1,2,4 and 6 weeks and extensively characterized by immunohistochemistry. In situ hybridization for X and Y chromosomes was conducted to identify female iPSCs and male fibroblasts in the RhGs. iPSC-RhGs showed increased epithelial thickening,rete ridge formation,increased cell proliferation and normalized expression of differentiation markers (keratins,involucrin,loricrin,SKALP/elafin) compared to standard RhGs,resulting in an epithelial phenotype very similar to the native gingiva. An increase in apoptotic cells was detected in iPSC-RhGs after 1 week air-exposed culture,and no iPSCs were detected in the hydrogels after 2 weeks air-exposed culture. The increase in apoptotic iPSCs after 1 week air-exposed culture correlated with an increase in keratinocyte proliferation responsible for the superior phenotype observed at 2 weeks. View Publication

Autoimmune uveitis is a leading cause of severe vision loss,and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq,RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU),revealing that EAU causes broad retinal neuron degeneration and marker downregulation,and that Müller glia may act as antigen-presenting cells. Moreover,EAU immune response is primarily driven by Th1 cells,and results in dramatic upregulation of CC chemokines,especially CCL5,in the EAU retina. Accordingly,overexpression of CCR5,a CCL5 receptor,in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU,by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together,our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03134-3. View Publication