参考文献

Human primordial germ cell-like cells (hPGCLCs) can be specified from human-induced pluripotent stem cells (hiPSCs),offering a valuable model for human germ cell development. However,further maturation steps of hPGCLCs rely on mouse feeders,or co-culture with mouse gonadal somatic cells. Exposure of hPGCLCs to human cellular niche has not been attempted. Here,we co-cultured female hPGCLCs in two distinct niche compartments. In reconstituted fetal ovary (rOv) culture,human fetal germ cells proliferated and initiated meiosis,while hPGCLCs upregulated gonadal germ cell markers such as DDX4. Additionally,hPGCLCs were supported and matured into migratory hPGCLCs in 3D co-culture with amnion-like cells (AMLCs). Compared to rOv,hPGCLCs in PGCLC/AMLC aggregates were less prone to dedifferentiate. In both niches,stem cell factor (SCF) was crucial for the survival of hPGCLCs. Together,this work underscores that a shift in niche is required for the further germ cell development of hPGCLCs. Graphical abstract Highlights•Reconstituted human fetal ovaries (rOvs) support meiosis entry of fetal germ cells•The rOvs support hPGCLCs to upregulate gonadal germ cell markers•hPGCLCs show less dedifferentiation in amnion-like cell aggregates compared to rOvs•SCF is not required for survival of fetal germ cells,but crucial for hPGCLCs Chuva de Sousa Lopes and colleagues used in vitro reconstituted human fetal ovaries (rOvs) as cellular niche to mature human primordial germ cell-like cells (hPGCLCs). hPGCLCs in rOvs matured but were prone to dedifferentiate. By contrast,hPGCLCs cultured with amnion-like cells were maintained without dedifferentiation and acquired migratory fate. In both systems,SCF was crucial for the survival of hPGCLCs. View Publication

Background and Objectives: Recent studies have identified variants in the kinesin family member 5A (KIF5A) gene that predispose to amyotrophic lateral sclerosis (ALS). These ALS-linked KIF5A variants lead to the exclusion of exon 27,resulting in the production of a mutated protein with an altered C-terminal region (KIF5A ΔExon27). Through whole genome sequencing,we identified a novel KIF5A intronic variant,rs1057522322 (c.2993-6C > A; chr12:57582596C > A,GRCh38.p14),in a family segregating ALS. Our goal is to investigate the effect of this variant on exon 27 splicing and to assess its functional consequences on KIF5A-mediated cargo transport. Methods: Induced pluripotent stem cells (iPSCs) were generated from siblings with and without the c.2993-6C > A variant. RT-PCR was performed on RNA extracted from iPSC-derived neurons to assess exon 27 splicing. Functional studies were conducted on iPSC-derived motor neurons (MNs). Results: RT-PCR confirmed that the c.2993-6C > A variant induced exon 27 skipping in KIF5A. Immunofluorescent staining showed that KIF5A ΔExon27 abolished the axonal interaction with splicing factor proline- and glutamine-rich,a cargo specifically transported by KIF5A. Under stress conditions,MNs carrying the c.2993-6C > A variant exhibited TDP-43 proteinopathy. Discussion: KIF5A intronic variant c.2993-6C > A could be a risk factor for ALS. KIF5A ΔExon27 impairs KIF5A-mediated cargo transport and contributes to ALS pathogenesis in a TDP-43–dependent manner. View Publication

Understanding of the factors governing immune responses in cancer remains incomplete,limiting patient benefit. In this study,we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time,with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges,including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context,whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection,and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade,revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment. View Publication

Bioresorbable nanopatterned scaffolds functionalized with polydopamine (PDA) and graphene oxide (GO) have been shown to promote the differentiation of murine neural stem cells (mNSCs) toward neural and glial lineages. Herein,we aim to evaluate the compatibility of these scaffolds for the culture and differentiation of both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Our results indicate that PDA and GO scaffolds support the topographic alignment of hESCs and hiPSCs cultures,while preserving their pluripotency characteristics. Upon differentiation,PDA and GO scaffolds guide cell specification toward the neuroectoderm germ layer and the neural crest. This promotes enhanced differentiation into both neural and supportive glial cells of the central nervous system (CNS),as well as Schwann cells of the peripheral nervous system (PNS). Moreover,nanopatterned scaffolds also support the differentiation of hESCs and hiPSCs toward endothelial precursors. These findings establish a novel culture platform that enables combined differentiation pathways,potentially relevant for applications in personalized medicine and regenerative cell therapy. View Publication

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis‐inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12‐abundant reticular (CAR) progenitor cells,resulting in loss of their hematopoiesis‐support and upregulation of osteogenic and pro‐apoptotic programs. In turn,peritrabecular osteolineage cells (OLCs) are activated in an injury‐specific,Wnt‐dependent manner,comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation,resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal β‐catenin expression is linked to fibrosis in patients,and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis,possibly being a clinically relevant therapeutic target. View Publication

Gestational transfer of brain‐reactive antibodies is a risk factor for neurodevelopmental disorders. Contactin‐associated protein‐like 2 (CASPR2) is a known target for pathogenic maternal autoantibodies which have been proposed to interfere with fetal neurodevelopment. However,the impact of CASPR2 antibodies on human brain development remains largely unknown. Here,to better understand the neurophysiological changes that occur in the presence of these pathogenic autoantibodies,we cultured unguided human neural organoids for a period of 6‐months in media containing anti‐CASPR2 antibodies. We then performed neurophysiological characterization via whole‐cell patch‐clamp and calcium imaging in acute organoid slices. Our results reveal that CASPR2 antibody exposure increased spontaneous synaptic activity,enhanced the maximal frequency of action potential firing and of spontaneous network activity. These findings are consistent with a state of neuronal hyperexcitability,a phenotype which is observed in several models of neurodevelopmental disorders. Mechanistically,the alterations observed in action potential waveform are in accordance with a role for CASPR2 in the regulation of voltage‐gated potassium channels and a pathological role for CASPR2 autoantibodies in driving neuronal hyperexcitability. Maternal antibodies targeting CASPR2 are a known risk factor for neurodevelopmental disorders,yet their impact on early human brain development remains unclear. We modeled this exposure using human neural organoids treated with patient‐derived CASPR2 antibodies up to the age of 6 months. Our study reveals that these antibodies drive neurons into a state of pathological hyperexcitability by specifically impairing action potential repolarization and enhancing excitatory synaptic transmission. These findings provide novel mechanistic evidence linking maternal autoimmunity to the excitation/inhibition imbalance characteristic of autism,highlighting a potential biological origin for antibody‐mediated neurodevelopmental conditions. View Publication

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior,drug response,and cell–matrix interactions. Here,we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates,facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation,GelMA preparation,bioprinting,and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research,including studies of metastasis,drug resistance,cancer cell lipid metabolism,and TME-associated hypoxia. View Publication

Myocardial infarction (MI) is a leading cause of death globally. Following MI,the heart undergoes remodeling leading to heart failure. The Hippo pathway is a major regulator of cell growth and survival in cardiomyocytes. Here,we show that serotonin receptor 2B (5-HT2B) regulates the Hippo pathway in cardiomyocytes and modulates heart remodeling following MI. 5-HT2B expression significantly enhanced the Hippo pathway effector Yes-associated protein (YAP) activity resulting in increased cardiomyocyte proliferation and decreased apoptosis. However,transgenic mice overexpressing 5-HT2B in cardiomyocytes had a lower survival rate post-MI. Conversely,modified mRNA (modRNA)-mediated transient 5-HT2B expression in the heart was sufficient to inhibit post-MI remodeling. Pharmacological screening of serotonergic compounds identified SB204741 as a modulator of the Hippo/YAP pathway in cardiomyocytes. SB204741 has been shown to protect the heart from adverse remodeling post-MI. Our findings identify 5-HT2B as a regulator of the Hippo pathway that can be targeted to improve cardiac phenotype following MI. Highlights•Serotonin receptor 2B (5HT2B) regulates the Hippo pathway and activates YAP in cardiomyocytes•5HT2B promotes cardiomyocyte proliferation and survival in vitro•Transgenic overexpression of 5HT2B exacerbates hypertrophic remodeling following MI•modRNA delivery of exogenous 5HT2B was partially protective against MI-induced remodeling Biochemical mechanism; Human metabolism; Molecular network View Publication

Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies,particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease,a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however,neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350,which binds complement receptors,and gp42,which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human,limiting clinical use. Using a transgenic mouse model,we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge,while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Graphical abstract Highlights•Transgenic mice were used to make genetically human EBV mAbs against gp350 and gp42•mAbs potently neutralize EBV infection by blocking receptor-ligand interactions•mAbs prevent EBV infection following EBV challenge in humanized mice Epstein-Barr virus (EBV) can cause serious illness,including cancer,especially in immunocompromised patients. There are no EBV-specific treatments. Chhan et al. leverage a transgenic mouse model to develop human monoclonal antibodies that block EBV entry. These antibodies prevent EBV infection in a murine challenge model offering hope for new therapies. View Publication

The immunomodulatory function of the gastric microbiota in cancer is poorly understood,partly due to the stomach’s acidic environment and limited microbial colonization. Here,by analyzing 68 paired human gastric cancer (GC) samples,we identify Ligilactobacillus salivarius as a commensal bacterium depleted in tumors but enriched in immune checkpoint blockade (ICB) responders. Oral administration of L. salivarius enhances anti-PD-1 efficacy in multiple GC mouse models by promoting pro-inflammatory macrophage activation. Mechanistically,bacterial extracellular vesicles (bEVs) derived from L. salivarius deliver 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) to tumors,where it activates formyl peptide receptor 1 (FPR1) on macrophages,triggering mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling. Moreover,2,3-BdpM augments the cytotoxic activity of chimeric antigen receptor (CAR)-Claudin18.2+ macrophages in an FPR1-dependent manner. These findings describe a microbial-macrophage axis that enhances GC immunotherapy and highlights the translational potential of orally deliverable microbial adjuvants. Graphical abstract Highlights•Intratumoral reduction of L. salivarius correlates with GC immunotherapy efficacy•bEVs derived from L. salivarius enhance immunotherapy efficacy in GC mouse models•2,3-BdpM in bEV triggers pro-inflammatory macrophage remodeling via FPR1•The cytotoxicity of CAR-Claudin18.2+ macrophages was amplified with 2,3-BdpM alone Yu et al. identify Ligilactobacillus salivarius as a gastric commensal enriched in immunotherapy responders. Oral administration enhances anti-PD-1 efficacy by delivering 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) via bacterial extracellular vesicles (bEVs) to activate pro-inflammatory macrophages through formyl peptide receptor 1 (FPR1),revealing a microbial-macrophage axis that potentiates gastric cancer immunotherapy. View Publication