技术资料

Regulatory T cells are a class of T lymphocytes which respond to activation signals by expanding their cell numbers,and whose culturing and expansion are of significant clinical interest. Cellular activation states are used to inform process control decisions such as restimulation and can be probed with experimental measurements of cell surface markers. However,these measurements are expensive,time-consuming,and invasive,and an urgent need exists for devising a non-invasive method for activation state monitoring that could be deployed on-line. Raman spectroscopy is a label-free and information-rich optical method that,when coupled to data analytical methods,can ameliorate these experimental issues. In this work,we quantitatively estimated experimental measurements of regulatory T cell activation markers with high accuracy. We simulated a clinical manufacturing setting by building an L1-regularized least-squares model with spectroscopic data from six regulatory T cell donors. Then,we validated the constructed model by accurately estimating different experimental measurements of biomarker values from two external donors,unseen by the model. We have devised a robust program to effectively estimate the activation state of regulatory T cells. We anticipate our method to be used with on-line Raman probes integrated into cell manufacturing devices for label-free monitoring of these processes. 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 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

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 high burden of dilated cardiomyopathy (DCM) in individuals of African descent remains incompletely explained. Here,to explore a genetic basis,we conducted a genome-wide association study in 1,802 DCM cases and 93,804 controls of African genetic ancestry (AFR). A nonsense variant (rs3211938:G) in CD36 was associated with increased risk of DCM. This variant,believed to be under positive selection due to a protective role in malaria resistance,is present in 17% of AFR individuals but <0.1% of European genetic ancestry (EUR) individuals. Homozygotes for the risk allele,who comprise ~1% of the AFR population,had approximately threefold higher odds of DCM. Among those without clinical cardiomyopathy,homozygotes exhibited an 8% absolute reduction in left ventricular ejection fraction. In AFR,the DCM population attributable fraction for the CD36 variant was 8.1%. This single variant accounted for approximately 20% of the excess DCM risk in individuals of AFR compared to those of EUR. Experiments in human induced pluripotent stem cell-derived cardiomyocytes demonstrated that CD36 loss of function impairs fatty acid uptake and disrupts cardiac metabolism and contractility. These findings implicate CD36 loss of function and suboptimal myocardial energetics as a prevalent cause of DCM in individuals of African descent. Genome-wide analysis in individuals of African ancestry identifies a nonsense variant in CD36 associated with increased risk of dilated cardiomyopathy (DCM),partly accounting for the higher incidence of DCM in African-ancestry populations. View Publication

Radiotherapy is a standard treatment of pediatric brain tumors. Though the survival rate has improved for many tumor types,most patients suffer long-term cognitive decline and there is currently no way of preventing radiation-induced damage to healthy brain tissue. Here,we used a human forebrain organoid model to investigate if the α2-adrenoceptor and I1-imidazoline receptor agonist clonidine could prevent radiotoxicity. We found that treatment of organoids with clonidine significantly reduced radiation-induced loss of neural progenitor cells,neurons,astrocytes,and oligodendrocyte lineage cells. Moreover,clonidine reduced overall DNA damage and signs of reactive gliosis in organoids. Our findings demonstrate that pharmacological rescue of radiation neurotoxicity is possible in a human brain organoid model and provides a rationale for future drug repurposing studies aiming to prevent radiation-induced brain injury in children treated with radiotherapy. 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

Management of chronic kidney disease (CKD) remains a major challenge due limited therapeutic options to reverse fibrosis,which is a critical feature in CKD. Partial epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is a key driver of fibrosis,and has become an important focus for kidney protection strategies. Blood platelets,a major source of circulating transforming growth factor beta (TGF-β),are implicated in pathogenesis of CKD,but their involvement in EMT and kidney fibrosis remains uncertain. Methods: We used two mouse models of renal fibrosis—diabetic kidney disease (DKD) and unilateral ureter obstruction (UUO)—to examine the connection between platelets,partial EMT,and fibrosis. Platelet inhibition or depletion was performed to assess EMT,cell cycle arrest,and fibrosis. In vitro,platelets were applied to TECs and kidney organoids. To determine the role of TGF-β signaling,we used TGF-βRI inhibitor. Expression of EMT,and fibrosis markers,as well as TGF-β1 signaling,were analyzed using western blot,reverse transcription quantitative PCR (RT-qPCR),enzyme-linked immunosorbent assay (ELISA),and immunostaining. Results: In both animal models,platelet inhibition or depletion resulted in reduced expression of cell cycle arrest marker p21,partial EMT and fibrosis. In vitro,activated platelets stimulated cell cycle arrest,EMT,and fibrosis in TECs and kidney organoids. Chronically injured TECs experience cell-cycle arrest which promote a paracrine EMT program in TECs,jointly leading to fibrosis. This platelet-mediated effect on cell cycle arrest and EMT was driven by TGF-β1 signaling,as selective inhibition of the TGF-β receptor rescued these dysfunctional phenotypes. Conclusions: Our study demonstrates that platelets activate the TGF-β1 pathway,leading to cell cycle arrest,EMT and renal fibrosis. These findings suggest that antiplatelet therapies may have potential renoprotective effects by protecting tubular homeostasis,attenuating partial EMT and fibrosis. View Publication

Bipolar disorder (BD) is a complex psychiatric condition usually requiring long-term treatment. Lithium (Li) remains the most effective mood stabilizer for BD,yet it benefits only a subset of patients,and its precise mechanism of action remains elusive. Exome sequencing has identified AKAP11 (A-kinase anchoring protein 11) as a shared risk gene for BD and schizophrenia (SCZ). Given that both the AKAP11-Protein Kinase A (PKA) complex and Li target and inhibit Glycogen Synthase Kinase-3 beta (GSK3β),we hypothesize that Li may partially normalize the transcriptomic and/or epigenomic alterations observed in heterozygous AKAP11-knockout (Het-AKAP11-KO) iPSC-derived neurons. In this study,we employed genome-wide approaches to assess the effects of Li on the transcriptome and epigenome of human iPSC-derived Het-AKAP11-KO neuronal culture. We show that chronic Li treatment in this cellular model upregulates key pathways that were initially downregulated by Het-AKAP11-KO,several of which have also been reported as downregulated in synapses of BD and SCZ post-mortem brain tissues. Moreover,we demonstrated that Li treatment partially rescues certain transcriptomic alterations resulting from Het-AKAP11-KO,bringing them closer to the WT state. We suggest two possible mechanisms underlying these transcriptomic effects: (1) Li modulates histone H3K27ac levels at intergenic and intronic enhancers,influencing enhancer activity and transcription factor binding,and (2) Li enhances GSK3β serine 9 phosphorylation,impacting WNT/β-catenin signaling and downstream transcription. These findings underscore Li’s potential as a therapeutic agent for BD and SCZ patients carrying AKAP11 loss-of-function variants or exhibiting similar pathway alterations to those observed in Het-AKAP11-KO models. View Publication

The lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-enhanced green fluorescent protein (eGFP) reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs,investigate their features,and facilitate their applications. Methods: hiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages,followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs. Results: hiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulated the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP-negative (-) cells,VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover,the dynamic expression of 394 known CD biomarkers in VSX2-eGFP+ cells at distinct developmental stages was analyzed herein for the first time. One CD biomarker,TNFRSF1B,which has never been reported to be expressed in RPCs,was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs. Conclusions: This study provides valuable insights into the molecular and cellular characteristics of human RPCs,especially their expression profiles of CD biomarkers,laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs. 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

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