技术资料

Bats can host viruses of pandemic concern without developing disease. The mechanisms underlying their exceptional resilience to viral infections are largely unresolved,necessitating the development of physiologically relevant and genetically tractable research models. Here,we developed respiratory and intestinal organoids that recapitulated the cellular diversity of the in vivo epithelium present in Rousettus aegyptiacus,the natural reservoir for the highly pathogenic Marburg virus (MARV). In contrast to human counterparts,bat organoids and mucosal tissue exhibited elevated constitutive expression of innate immune effectors,including type I interferon-ε (IFNε) and IFN-stimulated genes (ISGs). Upon infection with diverse zoonotic viruses,including MARV,bat organoids strongly induced type I and III IFN responses,which conferred robust antiviral protection. Type III IFNλ3 additionally displayed virus-independent self-amplification,acting as an ISG to enhance antiviral immunity. Our organoid platform reveals key features of bat epithelial antiviral immunity that may inform therapeutic strategies for viral disease resilience. Subject terms: Mucosal immunology,Viral infection View Publication

The intestinal epithelium of human infants is developmentally immature compared to that of adults. Exactly how this immaturity affects key epithelial functions and their interactions with nearby immune cells remains an understudied area of research,partly due to limited access to non-diseased infant gut tissues. Human intestinal organoids,or “mini guts” generated from tissue stem cells,are promising models for investigating intestinal biology and disease mechanisms. These three-dimensional structures closely mimic their tissue of origin,including cellular physiology and genetics. We have also previously shown that neonatal Th17 cells represent a distinct cell population with a cytokine profile skewed toward IL-22 production rather than IL-17A,as seen in adult Th17 cells. In this study,we sought to model the impact of neonatal-derived Th17 cytokine,namely IL-22 and the intestinal epithelium using infant-derived ileal enteroids. We generated enteroids from ileal biopsies from infants (< 6 months old) and cultured them for seven days with standard organoid growth media,organoid media supplemented with conditioned media from cord-blood-derived Th17 cells,or media supplemented with recombinant IL-22. We assessed morphological changes and conducted transcriptomics profiling via RNAseq. Exposing enteroids to neonatal Th17-cells-derived conditioned media led to enhanced growth,maturation,and differentiation as compared to control media. These effects were ablated when an IL-22 neutralizing antibody was used,while conversely,supplementing with recombinant IL-22 mimicked the Th17 effects,increasing intestinal epithelial cell proliferation and inducing marked differentiation of secretory cells. Our transcriptomic profiling similarly demonstrated significant changes in response to IL-22 with downregulation of Wnt and Notch signaling and upregulation of immune pathways,particularly interferon signaling. The transcriptomic data also suggested that IL-22 treatment led to changes in cell type composition with an increase in stem- and progenitor cells at the expense of enterocytes. Taken together,our data suggests that early-life intestinal development is likely influenced by IL-22-dependent crosstalk between the infant epithelium and exposure to neighboring Th17 cells. This promotes epithelial cell maturation and immune readiness,reflected at both the morphological and molecular levels. Our work also provides a relevant framework for studying healthy infant gut development,which can be further leveraged to examine early-life gastrointestinal disorders,model complex human disease,and therapeutic testing while reducing reliance on animal models. View Publication

Hematologic adverse events are common dose-limiting toxicities in drug development. Classical animal models for preclinical safety assessment of immunotherapies are often limited due to insufficient cross-reactivity with non-human homologous proteins,immune system differences,and ethical considerations. Therefore,we evaluate a human bone marrow (BM) microphysiological system (MPS) for its ability to predict expected hematopoietic liabilities of immunotherapeutics. The BM-MPS consists of a closed microfluidic circuit containing a ceramic scaffold covered with human mesenchymal stromal cells and populated with human BM-derived CD34+ cells in chemically defined growth factor-enriched media. The model supports on-chip differentiation of erythroid,myeloid and NK cells from CD34+ cells over 31 days. The hematopoietic lineage balance and output is responsive to pro-inflammatory factors and cytokines. Treatment with a transferrin receptor-targeting IgG1 antibody results in inhibition of on-chip erythropoiesis. The immunocompetence of the chip is established by the addition of peripheral blood T cells in a fully autologous setup. Treatment with T cell bispecific antibodies induces T cell activation and target cell killing consistent with expected on-target off-tumor toxicities. In conclusion,this study provides a proof-of-concept that this BM-MPS is applicable for in vitro hematopoietic safety profiling of immunotherapeutics. Subject terms: Biologics,Haematopoiesis,Lab-on-a-chip,Drug safety View Publication

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that influences several biological processes. Specific features in messenger RNAs (mRNAs) have been found to trigger decay by NMD,leading to the assumption that NMD sensitivity is an intrinsic quality of a given transcript. Here,we provide evidence that,instead,an overriding factor dictating NMD sensitivity is the cell environment. Using several genome-wide techniques to detect NMD-target mRNAs,we find that hundreds of mRNAs are sensitized to NMD as human embryonic stem cells progress to form neural progenitor cells. Another class of mRNAs escape from NMD during this developmental progression. We show that the differential sensitivity to NMD extends to in vivo scenarios,and that the RNA-binding protein,HNRNPL,has a role in cell type-specific NMD. We also addressed another issue in the field—whether NMD factors are core or branch-specific in their action. Surprisingly,we found that UPF3B,an NMD factor critical for the nervous system,shares only 30% of NMD-target transcripts with the core NMD factor UPF2. Together,our findings have implications for how NMD is defined and measured,how NMD acts in different biological contexts,and how different NMD branches influence human diseases. View Publication

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously,we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice,respectively. In this study,we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system,targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion,and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally,MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture,MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction,pro-oxidant profile amelioration,mitochondrial mass recovery,and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. View Publication

The tumor microenvironment and healing wounds both contain extremely high concentrations of adenosine triphosphate (ATP) compared to normal tissue. The P2Y2 receptor,an ATP-activated purinergic receptor,is typically associated with pulmonary,endothelial,and neurological cell signaling. Here,we examine ATP-dependent signaling in breast epithelial cells and how it is altered in metastatic breast cancer. Using rapid imaging techniques,we show how ATP-activated P2Y2 signaling causes an increase in intracellular Ca 2+ in non-tumorigenic breast epithelial cells,approximately 3-fold higher than their tumorigenic and metastatic counterparts. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to the cell edges after phalloidin staining,while the metastatic cells remain unaffected. The increase in intracellular Ca 2+ after ATP stimulation was blunted to control levels using a P2Y2 antagonist,which also prevented actin mobilization and significantly increased cell dissemination from spheroids in non-tumorigenic cells. Furthermore,the lack of Ca 2+ changes and actin mobilization in metastatic breast cancer cells could be due to the reduced P2Y2 expression,which correlates with poorer overall survival in breast cancer patients. This study elucidates the rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. View Publication

Acute myeloid leukemia (AML) is associated with unfavorable patient outcomes primarily related to disease relapse. Since specific types of leukemic hematopoietic stem and progenitor cells (HSPCs) are suggested to contribute to AML propagation,this study aimed to identify and explore relapse-initiating HSPC subpopulations present at diagnosis,using single-cell analysis (SCA). We developed unique high-resolution techniques capable of tracking single-HSPC-derived subclones during AML evolution. Each subclone was evaluated for chemo-resistance,in vivo leukemogenic potential,mutational profile,and the cell of origin. In BM samples of 15 AML patients,GMP-like and MLP-like HSPC subpopulations were identified as prevalent at relapse,exhibiting chemo-resistance to commonly used chemotherapy agents cytosine arabinoside (Ara-C) and daunorubicin. Reconstruction of phylogenetic lineage trees combined with genetic analysis of single HSPCs and single-HSPC-derived subclones demonstrated two distinct clusters,originating from MLP-like or GMP-like subpopulations,observed both at diagnosis and relapse. These subpopulations induced leukemia development ex vivo and in vivo. Genetic SCA showed that these relapse-related subpopulations harbored mutated EZH2 and TP53,detected already at diagnosis. This study,using combined molecular,functional,and genomic analyses at the level of single cells,identified patient-specific chemo-resistant HSPC subpopulations at the time of diagnosis,promoting AML relapse. View Publication

This study investigates the use of silver nanoparticles as a potential new treatment for colorectal cancer. Colorectal cancer is one of the most common cancers worldwide,and finding more effective treatments is essential. The researchers tested silver nanoparticles AgNPs with two different surface coatings to see how they affect cancer cells compared to healthy cells. One type of nanoparticles showed significant effects,reducing cancer cell growth and inducing cell death,while the other had minimal impact. These findings suggest that modifying the surface of nanoparticles could help target cancer cells more specifically,leading to treatments that are both more effective and have fewer side effects. This research could pave the way for new therapies for colorectal cancer and other types of cancer,ultimately improving patient outcomes and advancing cancer treatment strategies. View Publication

Malaria elimination faces challenges from drug resistance,stemming from mutations within the parasite’s genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature’s approach,and implementing methodologies to render indispensable host proteins inactive,holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain,which shares consensus sequence with the zinc endopeptidase family,possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor,BEL-A,we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally,thiorphan,a metallo-endopeptidase inhibitor,which specifically inhibits Kell activity,inhibited Plasmodium infection at nanomolar concentrations. Interestingly,individuals in malaria-endemic regions exhibit low Kell expression and activity,indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril,demonstrated potent antimalarial activity in vivo,highlighting Kell’s protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic. Subject terms: Parasite biology,Parasite host response View Publication

The in vitro generation of human red blood cells (RBCs) from stem cells,such as induced pluripotent stem cells (iPSCs),holds promise for transfusable RBCs but faces challenges,including RBC maturation,enucleation,and large-scale production. In this study,we evaluated the effect of conditional TAL1 overexpression on in vitro RBC production via hematopoietic cell-forming complex (HCFC) formation from iPSCs because TAL1 is a key regulatory transcription factor essential for erythropoiesis. TAL1 overexpression in iPSCs,either before or after hematopoietic induction,significantly enhanced HCFC formation and hematopoietic differentiation,as evidenced by increased hematopoiesis-related gene expression,a higher yield of glycophorin A (GPA)+/CD71+ cells,and elevated gamma hemoglobin levels. These findings highlight the potential of TAL1 as a powerful regulator of erythropoiesis in vitro and offer a promising strategy for improving RBC production from stem cells. However,the reduced enucleation efficiency observed after TAL1 overexpression indicates a key challenge that must be addressed to optimize the generation of fully functional,transfusable RBCs. Further research is required to balance the benefits of enhanced differentiation with the need for efficient enucleation,which is critical for the production of mature,viable RBCs. View Publication