技术资料

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

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

Parechovirus ahumpari 3 (HPeV-3) is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However,the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids,we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially,neuronal excitotoxicity. Elevated levels of extracellular glutamate,lactate dehydrogenase (LDH),and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions. View Publication

Antibody–drug conjugates (ADCs) represent a promising strategy in cancer therapy,enabling the targeted delivery of cytotoxic agents to tumor cells. In this study,we developed and characterized novel ADCs combining the anti-EGFR monoclonal therapeutic antibody Cetuximab (Cet) with two aminobisphosphonates (N-BPs) analogues of zoledronic acid (ZA): DC310 and the aminothiazole DC315. These conjugates aim to enhance antitumor efficacy of Cet in colorectal cancer (CRC) by both directly inhibiting tumor cell growth and activating Vδ2 T lymphocytes. We optimized the drug-antibody ratio (DAR),achieving significantly higher DARs compared to previously reported Cet-ZA conjugate,particularly with Cet-DC315 (DAR ≈ 23). Both ADCs retained selective EGFR binding in CRC cell lines and patient-derived organoids (PDO). Functionally,Cet-DC315 markedly inhibited proliferation of EGFR⁺ CRC cell lines in conventional cultures and 3D spheroids. Furthermore,Cet-DC-315 uniquely induced expansion and cytotoxic activation of Vδ2 T cells in co-cultures with CRC cell lines,PDO,and primary tumor samples. These findings suggest that ADCs incorporating novel N-BPs such as DC315 represent a potent approach for dual antitumor targeting through direct cytostatic effects and immune activation,offering a potential therapeutic advantage in the treatment of EGFR+ colorectal cancer. 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

Circulating tumor cell (CTC) detection in hepatocellular carcinoma (HCC) is limited not only by the rarity of CTCs but also by a heavy reliance on cell surface markers such as EpCAM,which are variably expressed or lost during tumor progression. Detecting intracellular markers,such as cytokeratin offers an important complementary and comprehensive strategy but remains technically limited in flow cytometry due to the need for fixation and permeabilization,which often lead to cell loss and surface epitope damage. In this study,we systematically evaluated the feasibility of using fixed samples for flow cytometry,using HepG2 cells,PBMCs,and CTCs from patients with HCC. Our results demonstrate that fixation enabled intracellular staining without compromising cell surface marker detection,even after short-term storage at 4 °C and long-term storage at -80 °C. Fixed samples,particularly fixed unfrozen,exhibited comparable staining performance to fresh samples with only a 7–10% reduction in cell recovery. Clinical validation in HCC patients confirmed successful CTC detection,and tumor-specific CTNNB1 mutations were identified in CTC-derived DNA but not in matched plasma cfDNA. These findings support fixed CTC sample workflows as a reliable and practical approach for CTC analysis in HCC. 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

Interleukin-1 receptor accessory protein (IL1RAP) is selectively expressed on both bulk blasts and leukemic stem cells (LSCs) in acute myeloid leukemia (AML),while its expression is virtually absent on normal hematopoietic stem cells (HSCs),making it an appealing target for chimeric antigen receptor (CAR) T cell therapy. Methods: We developed a novel IL1RAP-targeting CAR-T cells using a single-chain Fab (24scFab) fused to CD28 and CD3ζ costimulatory domains. CAR-T cells with a mutated IL1RAP-binding paratope were also generated as a control by introducing two point-mutations in the complementarity determining region (CDR) loops of the 24scFab domain. We tested the CAR-T cells in cell line-derived (CD) and patient-derived (PD) xenografts (X). To address persistence and activity of IL1RAP CAR-T cells,we then tested two approaches. First,we mutated two of the three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3ζ domain (i.e.,IL1RAP-1XX CAR-T). Second,we co-administered a synthetic miR-142 mimic (M-miR-142),previously shown to enhance T cell antileukemic activity,with IL1RAP CAR-T cells to AML xenografted mice. Results: IL1RAP CAR-T cells demonstrated a potent antileukemic activity in both AML CDX and PDX models. Target specificity was confirmed by the complete loss of function of IL1RAP-mutated CAR-T cells. IL1RAP-1XX CAR-T cells improved T cell persistence in vitro but failed to demonstrate therapeutic benefit compared with IL1RAP CAR-T cells in vivo. We previously reported that leukemic cell growth suppresses miR-142 biogenesis,thereby hindering the metabolic switch and impairing host T cell antileukemic activity; this was rescued by administration of M-miR-142. Thus,we hypothesized a similar impact of leukemic cells on CAR-T and that M-miR-142 treatment could rescue it and enhance the IL1RAP CAR-T cell antileukemic activity. We showed that both CDXs and PDXs receiving M-miR-142 and IL1RAP CAR-T lived significantly longer than those receiving scrambled oligonucleotide and IL1RAP CAR-T or mutated CAR-T controls (median survival of PDX: 78 vs 51 vs 24 days). Conclusions: We have identified a potentially novel strategy to enhance CAR-T cell persistence and efficacy in AML by counteracting a leukemia-induced,microRNA-deficiency mediated mechanism of immune suppression. View Publication

Effective therapies for Alzheimer’s disease (AD) remain to be developed. APOE4 is the strongest genetic risk factor for late-onset AD. Pericyte degeneration and blood–brain barrier (BBB) disruption are thought to be early biomarkers of AD and contribute to cognitive decline in APOE4 carriers,representing potential therapeutic targets. Our previous studies have shown that pericyte transplantation is one of the most effective strategies for BBB restoration,exhibiting great therapeutic potential for APOE4-related BBB damage and AD phenotypes. Methods: APOE4/4 mice were treated with pericytes derived from APOE3/3 human induced pluripotent stem cells (hiPSCs). Behavioral tests,AD pathologies,and BBB integrity were assessed. Subsequently,temporal and spatial distribution of the transplanted pericytes was analyzed using tdTomato+ lentivirus labeling. Next,therapeutic effects of apoptotic vesicles (ApoVs) generated from APOE3/3 pericytes were evaluated in APOE4/4 pericytes in vitro. Additionally,transcriptomic and proteomic profiling were performed to identify key effector molecules in pericyte-derived ApoVs. Finally,the therapeutic effects of ApoVs derived from pericytes were evaluated in APOE4/4 mice. Results: Early,multiple transplantations of pericytes derived from APOE3/3 hiPSCs robustly rescued cognitive decline and AD pathologies,restored BBB integrity,and prevented in situ pericyte degeneration in aged APOE4/4 mice. Intriguingly,ApoVs released from the infused cells,rather than the transplanted pericytes,were predominantly distributed in the brain,which were ingested by in situ APOE4/4 pericytes and then promoted functional recovery. We further characterized insulin growth factor-2 (IGF-2) as a key factor in APOE3/3 pericyte-derived ApoVs. Infusion of the in vitro generated ApoVs from APOE3/3 pericytes demonstrated distinct therapeutic effects in APOE4/4 mice,which were reversed by IGF2 knockout. Conclusions: APOE3/3 pericytes or APOE3/3 pericyte-derived IGF2-rich ApoVs may offer promising therapeutic strategies for APOE4-associated AD. 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

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total,3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9,Collagen 18A,Tropomyosin 1,BASP1,RUVBL1,and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol. View Publication