技术资料

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication

MEF2C encodes a transcription factor that is critical in nervous system development. Here,to examine disease-associated functions of MEF2C in human microglia,we profiled microglia differentiated from isogenic MEF2C-haploinsufficient and MEF2C-knockout induced pluripotent stem cell lines. Complementary transcriptomic and functional analyses revealed that loss of MEF2C led to a hyperinflammatory phenotype with broad phagocytic impairment,lipid accumulation,lysosomal dysfunction and elevated basal inflammatory cytokine secretion. Genome-wide profiling of MEF2C-bound sites coupled with the active regulatory landscape enabled inference of its transcriptional functions and potential mechanisms for MEF2C-associated cellular functions. Transcriptomic and epigenetic approaches identified substantial overlap with idiopathic autism datasets,suggesting a broader role of human microglial MEF2C dysregulation in idiopathic autism. In a mouse xenotransplantation model,loss of MEF2C led to morphological,lysosomal and lipid abnormalities in human microglia in vivo. Together,these studies reveal mechanisms by which reduced microglial MEF2C could contribute to the development of neurological diseases. Coufal and colleagues generated microglia from human iPS cells to examine mechanistic roles of the transcription factor MEF2C and how these roles might relate to the autism phenotype seen following the loss of MEF2C in human microglia. View Publication

Respiratory organoids have emerged as a powerful in vitro model for studying respiratory diseases and drug discovery. However,the high-throughput analysis of organoid images remains a challenge due to the lack of automated and accurate segmentation tools. This study presents a semi-automatic algorithm for image analysis of respiratory organoids (nasal and lung organoids),employing the U-Net architecture and CellProfiler for organoids segmentation. The algorithm processes bright-field images acquired through z-stack fusion and stitching. The model demonstrated a high level of accuracy,as evidenced by an intersection-over-union metric (IoU) of 0.8856,F1-score = 0.937 and an accuracy of 0.9953. Applied to forskolin-induced swelling assays of lung organoids,the algorithm successfully quantified functional differences in Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-channel activity between healthy donor and cystic fibrosis patient-derived organoids,without fluorescent dyes. Additionally,an open-source dataset of 827 annotated respiratory organoid images was provided to facilitate further research. Our results demonstrate the potential of deep learning to enhance the efficiency and accuracy of high-throughput respiratory organoid analysis for future therapeutic screening applications. Author summaryIn this study,we developed a semi-automated tool to analyze images of respiratory organoids—3D cell structures that mimic the human respiratory system. These organoids are vital for studying diseases like cystic fibrosis and testing potential drugs,but manually analyzing their images is time-consuming and prone to errors. Our tool uses artificial intelligence (AI) to quickly and accurately measure organoid size and shape from bright-field microscope images,eliminating the need for fluorescent dyes that can harm cells. We trained our AI model on a publicly shared dataset of 827 annotated organoid images,achieving high accuracy in detecting and quantifying organoids. When applied to cystic fibrosis research,the tool successfully measured differences in organoid swelling (forskolin-induced swelling - a key test for drug response) between healthy and patient-derived samples. By making our dataset and method openly available,we hope to support further research into respiratory diseases. Our work bridges the gap between complex lab techniques and practical applications,offering a faster,more reliable way to study human health and disease. View Publication

A growing body of evidence implicates inflammation as a key hallmark in the pathophysiology of Parkinson’s disease (PD),with microglia playing a central role in mediating neuroinflammatory signaling in the brain. However,the molecular mechanisms linking microglial activation to dopaminergic neuron degeneration remain poorly understood. In this study,we investigated the contribution of the PD-associated LRRK2-G2019S mutation to microglial neurotoxicity using patient-derived induced pluripotent stem cell (iPSC) models. We found that LRRK2-G2019S mutant microglia exhibited elevated activation markers,enhanced phagocytic capacity,and increased secretion of pro-inflammatory cytokines such as TNF-α. These changes were associated with metabolic dysregulation,including upregulated glycolysis and impaired serine biosynthesis. In 3D midbrain organoids,these overactivated microglia resulted in dopaminergic neuron degeneration. Notably,treating LRRK2-G2019S microglia with oxamic acid,a glycolysis inhibitor,attenuated microglial inflammation and reduced neuronal loss. Our findings underscore the link between metabolic targeting in microglia and dopaminergic neuronal loss in LRRK2-G2019S mutation,and highlight a potential strategy that warrants further preclinical evaluation. View Publication

Chronic compressive cervical spinal cord injury (cCSCI),a debilitating condition,lacks effective treatment options. Addressing this gap,our study introduces a novel rat model of cCSCI developed through spinal cord compression via synthetic polyether sheet implantation,closely mimicking human pathology. We evaluated the model’s fidelity utilizing a comprehensive series of behavioral,electrophysiological,and histological assessments. Our research also explored the therapeutic potential of oligodendrogenic neural progenitor cells (oNPCs) derived from induced pluripotent stem cells. Transplanted oNPCs successfully integrated into the host spinal cord,differentiated into neurons,astrocytes,and oligodendrocytes,and demonstrated a remarkable capacity for enhancing neuroplasticity. Electrophysiological analyses revealed significant improvements in motor evoked potentials and a rectification of the excitability imbalance posttransplantation,indicating substantial recovery of motor circuits. Histological findings complemented these results,showing enhanced remyelination and a reduction in excitatory transmitter expression in the residual gray matter. Functionally,the transplantation of oNPCs led to marked improvements in grip strength,locomotor abilities,and sensory functions,surpassing those seen with standard treatments. This study not only provides a novel and reliable rat model of cCSCI for further research but also highlights the potential of oNPCs as a transformative approach for spinal cord injury therapy,suggesting their significant role in neural regeneration and repair. View Publication

BackgroundThe cycling intestinal stem cells (ISCs) exhibit radiosensitivity,and their death or impaired regenerative capacity following irradiation may result in intestinal barrier dysfunction. The cystathionine-β-synthase (CBS)/H2S axis plays a critical role in regulating cell proliferation,reactive oxygen species scavenging,and the DNA damage response. However,it remains unclear whether the CBS/H2S axis modulates ISC homeostasis and tissue radiosensitivity. Methods: Intestinal epithelium specific conditional CBS knockout mice were generated by crossing CBSfl/+ mice with Villin-CreERT2 mice. CAGGCre-ER™ mice were crossed with CBSfl/fl mice to achieve CBS knockout in multiple tissues and cell types. The Lgr5-Tdtaomato-Flag mice were generated by CRISPR/Cas9 system. The CBS inhibitor AOAA or the H2S donor GYY4137 was used to treat mice or intestinal crypt organoids. Hematoxylin and eosin,immunohistochemistry,immunofluorescence,Western blot,qRT-PCR,et al. were employed to investigate the role of the CBS/H2S axis in ISCs homeostasis and radiation-induced intestinal damage. Results: Lgr5 + ISCs and progenitor cells expressed higher levels of CBS than differentiated cells. The cecum and colon expressed significant higher CBS levels than the small intestine. Treatment with the H2S donor GYY4137 enhanced the proliferation of intestinal organoids in vitro,while inhibition of CBS by AOAA reduced this effect. Genetic knockout of CBS in the intestinal epithelium or global downregulation of CBS driven by CAGG-CreER™ in vivo did not affect ISC proliferation or differentiation under physiological conditions. Pharmacological regulation of the CBS/H2S axis in vitro failed to protect organoids from radiation-induced damage. Interestingly,administration of AOAA in vivo reduced radiation-induced atrophy of the intestinal mucosa. Furthermore,global downregulation of CBS significantly promoted ISC recovery after irradiation exposure. However,intestinal epithelium-specific CBS knockout did not confer radioprotective effects. Conclusions: Our findings suggest that the CBS/H2S axis contributes to the regulation of ISC homeostasis and represents a potential target for radiation protection,mediated through the intervention of non-epithelial cells. View Publication

The selective peroxisome proliferator–activated receptor delta (PPARD) agonist seladelpar reduces liver injury and modulates bile acid metabolism in preclinical models. Seladelpar was recently approved for the secondary treatment of primary biliary cholangitis (PBC). Despite its beneficial effects for liver diseases,the target cells of seladelpar on a single-cell level remain unknown. This study is aimed at investigating the effect of seladelpar on single liver cells. Methods and Results: CD-1 mice were gavaged with vehicle or seladelpar (10 mg/kg body weight),and the liver was harvested 6 h later. Single-nuclei RNA sequencing (snRNA-seq) analysis showed the engagement of PPARD target genes primarily in hepatocytes and cholangiocytes by seladelpar. The top two upregulated genes,Ehhadh and Cyp4a14,are related to fatty acid metabolism and were increased in hepatocytes,cholangiocytes,and Kupffer cells. Abcb4,an important canalicular transporter with hepatoprotective effects,was significantly upregulated in hepatocytes. We confirmed upregulated Abcb4 gene expression in seladelpar-treated primary mouse hepatocytes isolated from C57BL/6 mice. We further incubated nonparenchymal liver cells with seladelpar. Although there was a significant increase in the PPARD-responsive genes Pdk4 and Angptl4 in cholangiocytes,Kupffer cells,and hepatic stellate cells,seladelpar did not exert specific liver-protective effects in these cell types. Conclusions: The selective PPARD agonist seladelpar induced PPARD-responsive genes primarily in hepatocytes and cholangiocytes. Seladelpar upregulated Abcb4 in hepatocytes,which might contribute to its beneficial effects in cholestatic liver disorders. View Publication

USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP),a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans,and its earliest pathogenic events remain unknown. Here,we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A−/− induced pluripotent stem cells. Methods: We assessed organoids for cellular,molecular,and morphological defects using serial live imaging and whole organoid and fixed section analyses. Results: Both patient-derived and isogenic-USH2A−/− organoids showed preferential rod photoreceptor loss followed by widespread degeneration,consistent with the clinical phenotype. Additionally,isogenic-USH2A−/− organoids showed early defects in proliferation and structure. Conclusions: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP,and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features,we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies. View Publication

The maintenance of a basal immunoinflammatory signature in hematopoietic stem/progenitor cells (HSPCs) constitutes a fundamental regulatory axis governing hematopoietic competence and immune effector generation. While epigenetic repressors constrain this inflammatory phenotype,the molecular amplifiers that preserve this critical state remain undefined. Through integrated single-cell transcriptomic/epigenomic profiling and functional interrogation,we identify Setd2-mediated H3K36me3 as an indispensable epigenetic amplifier sustaining baseline inflammation in murine HSPCs. Setd2 ablation specifically eliminated interferon (IFN)-enriched HSPC subpopulations and attenuated inflammatory signaling cascades. Functionally,Setd2-deficient HSPCs exhibited impaired IFNγ responsiveness,compromised B-lymphopoiesis,and diminished reconstitution capacity due to Lin−c-Kit+Sca1high cell depletion. Paradoxically,Setd2 loss conferred resistance to IFNγ-induced HSPCs exhaustion,which may contribute to the maintenance of Setd2-deficient HSPCs in our myelodysplastic syndrome (MDS) model under the inflammatory milieu. Mechanistically,Setd2 sustained chromatin accessibility and enhancer (H3K27ac) activity at inflammatory gene loci. This work delineates a critical link between Setd2-mediated chromatin regulation,baseline inflammation,HSPC function,and immune competence,providing insights into inflammatory dysregulation in hematopoietic malignancies like MDS. View Publication

Aggregated α-synuclein (αSyn) is a pathological hallmark of Parkinson’s disease (PD),yet other protein aggregates,including tau,are commonly observed in PD brains. This suggests that PD is not solely a synucleinopathy but may involve multiple,coexisting proteinopathies. Mutations in LRRK2,particularly the G2019S (GS),are the most common cause of familial PD. LRRK2-PD has been associated with both αSyn and tau pathology; however the mechanistic links between LRRK2 dysfunction and protein aggregation remain incompletely defined. Here we opted to investigate whether LRRK2 contributes to αSyn and tau pathology through common molecular pathways or via distinct cellular mechanisms. Viral vector-mediated αSyn overexpression in GS LRRK2 knock-in mice led to enhanced dopaminergic neurodegeneration,increased phosphorylated αSyn levels,pronounced neuroinflammation,and accumulation of lysosomal proteins,suggesting impaired αSyn clearance and immune activation as key drivers. Human iPSC-derived dopaminergic neurons from GS LRRK2 PD patients mirrored these findings. In contrast viral vector-mediated overexpression of tau in GS LRRK2 knock-in mice promoted tau phosphorylation but did not significantly affect neuroinflammation,lysosomal markers,or neurodegeneration,indicating a primarily cell-autonomous mechanism. Our results reveal a mechanistic divergence in how GS LRRK2 impacts αSyn and tau pathologies,supporting the notion that LRRK2 kinase activity contributes to PD pathogenesis through different pathways,thereby highlighting its potential as a therapeutic target in both familial and sporadic PD. View Publication

Osteoporosis (OP) could lead to the alteration of bone marrow microenvironment and non-homeostasis of hematopoiesis,which could increase the incidence of hematologic malignancies. However,whether myeloid-biased hematopoiesis occurred and contributed to the leukemogenesis under the condition of OP remains unclear. Results: This study successfully induced a mouse model for OP by hindlimb unloading,which shows increased myeloid cells and decreased B cells in the peripheral blood (PB). Furthermore,our study demonstrates that the myeloid-biased subset of HSPCs (hematopoietic stem and progenitor cells) with reduced differentiation and apoptosis,including multipotent progenitor 3 (MPP3) and granulocyte-monocyte progenitors (GMPs),were expanded in the OP mice. The expansion of myeloid-biased HSPCs contributes to the accumulation of HSPCs in the bone marrow and increased myeloid cells in the PB of OP mice. In the expanded pool of HSPCs,OP mice specifically enriched subsets were identified and profiled by single cell RNA-seq,including subHSCs from primitive HSCs,MPP3-1 from MPP3,GMP5 from GMPs,MkP2 from megakaryocyte progenitors and EryP1 from erythrocyte progenitors. Meanwhile,those OP-HU mice enriched subsets shared significantly up- and down-regulated genes enriched in chromatin modification and cell differentiation and apoptosis such as Bromodomain-containing protein 4 (Brd4),encoding an important chromatin remodeling protein,and Proteinase 3 (Prtn3). Moreover,the specific transcription factors corresponding to the expansion of subHSCs,MPP3-1,GMP5 and EryP1 in OP-HU mice were identified as Zfp951,Nfic,Maz and Ezh2. Finally,inhibition of BRD4 in vivo could partially restore the phenotype of OP-HU mice and the expression of genes regulating HSPC expansion,differentiation and apoptosis. Conclusions: First of all,our study shows that OP could induce the unbalanced hematopoiesis and enhances the myeloid-biased hematopoiesis. Secondly,OP mice enriched subsets of HSPCs were identified and characterized with enhanced chromatin remodeling,reduced differentiation and resistance to apoptosis. Finally,this study demonstrate that Brd4 regulated gene programs endow the myeloid-biased subsets of HSPCs with tumor cell-like characters in OP mice,which may increase the incidence of the leukemic evolution. This study sheds light on the importance for the prevention of myeloid leukemogenesis in human with OP. View Publication

Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens,including parasites. These ETs function to trap,immobilize,and eventually kill pathogens. In line with this,monocytes and macrophages have been shown to release ETs,known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii,this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods: Primary human and bovine monocytes,monocytic THP-1 cells,and THP-1 cell-derived macrophages (M0-,M1-,and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM),transmission electron microscopy (TEM),immunofluorescencemicroscopy,and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally,the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6,and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion: Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore,the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs,although this was a rare event. In primary human monocytes,an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS),primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells,THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS,all THP-1 cell-derived macrophages showed an enhanced release of IL-1β,and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent,MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites. View Publication