技术资料

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids,organoids,tumoroids,or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies,vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints,we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids,as well as blood vessel organoids generated from pluripotent stem cells,cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids,as they successfully provide intravascular perfusion to these structures. We find that organoid growth,maturation,and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics. Subject terms: Stem-cell biotechnology,Tissue engineering,Biomedical engineering,Induced pluripotent stem cells,Microfluidics View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS,accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic,which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach,we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila . Consistent with a conserved role in disease pathogenesis,pharmacological inhibition of both proteins rescued disease-relevant phenotypes,including mitochondrial trafficking defects and neuromuscular junction failure,in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies,mice,and human iPSC-sMNs,we observed reduced GSK3 inhibitory phosphorylation,suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore,we found that PP2A acts upstream of GSK3,affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs,and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover,increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity,confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers,and reveal an unexplored mechanistic link between PP2A,GSK3,and kinesin-1,that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease. The online version contains supplementary material available at 10.1007/s00401-024-02689-y. View Publication

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM),which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65–70%,patients with LM experience poor survival rates of only 19–30%. Unacceptably,this situation that has remained unchanged for 30 years. Thus,there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors,including 4 matched sets,were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy. Subject terms: Bone cancer,Sarcoma,Gene expression View Publication

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer’s disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology,we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein,we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM,however,these compounds lack selectivity and this limits their utility as chemical tools. View Publication

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations,maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However,patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here,we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model,we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity. Subject terms: Cancer stem cells,Acute myeloid leukaemia,Target validation View Publication

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies,including acute myeloid leukemia (AML). Here,we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this,we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78,CD123,or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus,we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations. View Publication

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers,MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK,ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability. View Publication

During kidney development,nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification,transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids,which recapitulate nephrogenesis,to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids,contrary to in vivo mouse studies,likely by activating a cell-adhesion program. While Wnt/β-catenin signaling specifies nephron fate,we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney,with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury. View Publication

A new perspective suggests that a dynamic bidirectional communication system,often referred to as the microbiome-gut-brain axis,exists among the gut,its microbiome,and the central nervous system (CNS). This system may influence brain health and various brain-related diseases,especially in the realms of neurodevelopmental and neurodegenerative conditions. However,the exact mechanism is not yet understood. Metabolites or extracellular vesicles derived from microbes in the gut have the capacity to traverse the intestinal epithelial barrier or blood–brain barrier,gaining access to the systemic circulation. This phenomenon can initiate the physiological responses that directly or indirectly impact the CNS and its function. However,reliable and controllable tools are required to demonstrate the causal effects of gut microbial-derived substances on neurogenesis and neurodegenerative diseases. The integration of microfluidics enhances scientific research by providing advanced in vitro engineering models. In this study,we investigated the impact of microbe-derived metabolites and exosomes on neurodevelopment and neurodegenerative disorders using human induced pluripotent stem cells (iPSCs)-derived neurons in a gut-brain axis chip. While strain-specific,our findings indicate that both microbial-derived metabolites and exosomes exert the significant effects on neural growth,maturation,and synaptic plasticity. Therefore,our results suggest that metabolites and exosomes derived from microbes hold promise as potential candidates and strategies for addressing neurodevelopmental and neurodegenerative disorders. View Publication

The peripheral immune system is important in neurodegenerative diseases,both in protecting and inflaming the brain,but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here,we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment ( n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11 C-PiB PET and 18 F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn,we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis. Subject terms: Protein aggregation,Neuroimmunology,Dementia View Publication

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally,the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells,a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction. We generated NOD1 −/− -Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow–retina axis. Elevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1 −/− -Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration,as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations,including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation,and the results further indicated that NOD1 had a protective effect against DR. Mechanistically,loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina,subsequently leading to diminished neutrophil chemoattraction and NETosis. The results of our study unveil,for the first time,the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation,culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR. The online version contains supplementary material available at 10.1186/s13287-024-03654-y. View Publication

Windham et al. discover that APOE in astrocytes can traffic to lipid droplets (LDs),where it modulates LD composition and size. Astrocytes expressing the Alzheimer’s risk variant APOE4 form large LDs with impaired turnover and increased peroxidation sensitivity. View Publication