技术资料

Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication

Brain organoids derived from human pluripotent stem cells (hPSCs) hold immense potential for modeling neurodevelopmental processes and disorders. However,their experimental variability and undefined organoid selection criteria for analysis hinder reproducibility. As part of the Bavarian ForInter consortium,we generated 72 brain organoids from distinct hPSC lines. We conducted a comprehensive analysis of their morphological and cellular characteristics at an early stage of their development. In our assessment,the Feret diameter emerged as a reliable,single parameter that characterizes brain organoid quality. Transcriptomic analysis of our organoid identified the abundance of unintended mesodermal differentiation as a major confounder of unguided brain organoid differentiation,correlating with Feret diameter. High-quality organoids consistently displayed a lower presence of mesenchymal cells. These findings provide a framework for enhancing brain organoid standardization and reproducibility,underscoring the need for morphological quality controls and considering the influence of mesenchymal cells on organoid-based modeling. Subject terms: Mesenchymal stem cells,Induced pluripotent stem cells,Stem-cell differentiation View Publication

Understanding diseases as the result of continuous transitions from a healthy system is more realistic than understanding them as discrete states. Here,we designed the spectrum formation approach (SFA),a machine learning-based method that extracts key features contributing to disease state continuity. We applied the SFA to transcriptomic data from patients with progressive liver disease and neurodegenerative movement disorders to examine its effectiveness in identifying biologically relevant gene sets. The SFA identified transcription factors that potentially regulate liver inflammation and voluntary movement. In neurodegenerative disorders,the SFA also identified genes regulated by ETS-1,with unclear effects on movement. In functional assessment using human iPSC-derived neurons,ETS-1 overexpression disrupted dopamine receptor balance,reduced GABA-producing enzyme levels,and promoted cell death. These findings suggest that the SFA enables the discovery of regulatory factors capable of modifying disease states and provides a framework for the continuity-based interpretation of biological systems. Subject terms: Diseases,Pathogenesis,Signs and symptoms View Publication

Variants of phospholipase C gamma 2 (PLCG2),a key microglial immune signaling protein,are genetically linked to Alzheimer's disease (AD) risk. Understanding how PLCG2 variants alter microglial function is critical for identifying mechanisms that drive neurodegeneration or resiliency in AD. Induced pluripotent stem cell (iPSC) –derived microglia carrying the protective PLCG2 P522R or risk‐conferring PLCG2 M28L variants,or loss of PLCG2,were generated to ascertain the impact on microglial transcriptome and function. Protective PLCG2 P522R microglia showed significant transcriptomic similarity to isogenic controls. In contrast,risk‐conferring PLCG2 M28L microglia shared similarities with PLCG2 KO microglia,with functionally reduced TREM2 expression,blunted inflammatory responses,and increased proliferation and cell death. Uniquely,PLCG2 P522R microglia showed elevated cytokine secretion after lipopolysaccharide (LPS) stimulation and were protected from apoptosis. These findings demonstrate that PLCG2 variants drive distinct microglia transcriptomes that influence microglial functional responses that could contribute to AD risk and protection. Targeting PLCG2‐mediated signaling may represent a powerful therapeutic strategy to modulate neuroinflammation. The impact of Alzheimer's disease protective‐ and risk‐associated variants of phospholipase C gamma 2 (PLCG2) on the transcriptome and function of induced pluripotent stem cell (iPSC) –derived microglia was investigated. PLCG2 risk variant microglia exhibited a basal transcriptional profile similar to PLCG2‐deficient microglia but significantly different from isotype control and the transcriptionally similar PLCG2 protective variant microglia. PLCG2 risk variant and PLCG2‐deficient microglia show decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2). The differential transcriptional pathways of protective and risk‐associated PLCG2 variant microglia functionally affect proliferation,apoptosis,and immune response. Protective PLCG2 microglia show resilience to apoptosis and increased cytokine/chemokine secretion upon exposure to lipopolysaccharide (LPS). View Publication

Alpha-synuclein (aSyn) post-translational modifications (PTM),especially phosphorylation at serine 129 and C-terminal truncations,are highly enriched in Lewy bodies (LB),Lewy neurites,and other pathological aggregates in Parkinson’s disease and synucleinopathies. However,the precise role of these PTM in pathology formation,neurodegeneration,and pathology spreading remains unclear. Here,we systematically investigated the role of post-fibrillization C-terminal aSyn truncations in regulating uptake,processing,seeding,and LB-like inclusion formation using a neuronal seeding model that recapitulates LB formation and neurodegeneration. We show that C-terminal cleavage of aSyn fibrils occurs rapidly post exogenous fibril internalization and during intracellular LB-like inclusion formation. Blocking cleavage of internalized fibrils does not affect seeding,but inhibiting enzymes such as calpains 1 and 2 alters LB-like inclusion formation. We show that C-terminal truncations,along with other PTMs,regulate fibril interactome remodeling,shortening,lateral association,and packing. These findings reveal distinct roles of C-terminal truncations at different aggregation stages on the pathway to LB formation,highlighting the need for consideration of stage‑specific strategies to target aSyn proteolytic cleavages. View Publication

The rare and rapidly progressive neurodegenerative disease multiple system atrophy (MSA) mainly affects the striatum and other subcortical brain regions. In this atypical Parkinsonian syndrome,the protein alpha-synuclein aggregates and misfolds in neurons as well as glial cells and is released in elevated amounts by hypoexcitable neurons. Mitochondrial dysregulation affects the biosynthesis of coenzyme Q10 and the activity of the respiratory chain,as shown in an induced pluripotent stem cell (iPSC) model. Proteome studies of cerebrospinal fluid and brain tissue from MSA patients yielded inconsistent results regarding possible protein changes due to small and combined groups of atypical Parkinsonian syndromes. In this study,we analysed the cellular proteome of MSA patient-derived striatal GABAergic medium spiny neurons. We observed 25 significantly upregulated and 16 significantly downregulated proteins in MSA cell lines compared to matched healthy controls. Various protein types involved in diverse molecular functions and cellular processes emphasise the multifaceted pathomechanisms of MSA. These data could contribute to the development of novel disease-modifying treatment strategies for MSA patients. View Publication

Hepatitis E virus (HEV) is a significant human pathogen causing both acute and chronic infections worldwide. The cell-intrinsic antiviral response serves as the initial defense against viruses and has been shown to be activated upon HEV infection. HEV can replicate in the presence of this response,but the underlying mechanisms remain poorly understood. Here,we investigated the roles of the structural proteins ORF2 and ORF3 in the cell-intrinsic antiviral response to HEV infection. Mechanistically,we validated that ectopic ORF2,but not ORF3,interfered with antiviral and inflammatory signaling downstream of pattern recognition receptors,in part through interaction with the central adaptor protein TANK binding kinase 1. In the full-length viral context,ORF2 contributed to a reduced antiviral response and consequently,more efficient viral replication. In addition,we discovered a protective mechanism mediated by ORF2 that shielded viral replication from antiviral effectors. Using single-cell RNA-sequencing,we confirmed that the presence of ORF2 in infected cells dampened antiviral responses in both actively infected cells and bystanders. As a consequence,we found that early in the infection process,the progression of authentic HEV infection relied on the presence of ORF2,facilitating a balance between viral replication and the antiviral response. Altogether,our findings shed new light on the multifaceted role of ORF2 in the HEV life cycle and improve our understanding of the determinants that contribute to persistent HEV replication in cell culture. Author summaryHepatitis E virus (HEV) is an important yet often underestimated pathogen. Depending on the genotype,HEV infections can progress to chronicity,but the underlying mechanisms remain poorly understood. To gain insight into potential determinants,we investigated how HEV evades the cell-intrinsic antiviral response. We discovered that the HEV capsid protein ORF2 is crucial in limiting this response by interfering with antiviral signaling pathways and shielding viral replication from immune effectors. This balance between viral replication and the antiviral response contributes to persistent HEV infection in cell culture. Our findings reveal a new role for the HEV capsid protein in the viral life cycle and highlight it as an important target for novel therapeutic approaches. View Publication

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. View Publication

Neuronal TDP-43 aggregates are a hallmark ALS pathology. The integrated stress response (ISR) occurs downstream of TDP-43 pathology and may promote neurodegeneration. Here we demonstrate that a CNS penetrant small molecule eIF2B activator inhibits the ISR in cellular models of ALS and the brain of an inducible mouse model of TDP-43 pathology,where it transiently slowed progression of locomotor deficits and neurodegeneration. ISR activation was observed in ALS patient spinal cord and CSF. The investigational drug DNL343 was advanced into Phase 1 and Phase 1b randomized,double-blind,placebo-controlled trials in healthy and ALS participants,respectively (NCT04268784/NCT05006352); the primary objective in both studies was to investigate the safety and tolerability DNL343. DNL343 demonstrated a half-life supporting once-daily dosing and showed extensive CSF distribution. DNL343 was generally well tolerated and reduced ISR biomarkers in peripheral blood mononuclear cells and CSF of ALS participants. Therefore,DNL343 is a useful investigational drug to explore the effects of ISR inhibition in ALS models and individuals with neurological diseases. Flores et al. show that brain-penetrant eIF2B agonists suppress ISR activation in cellular and mouse models of ALS and reduce ISR biomarkers in humans,enabling further clinical studies of ISR inhibition in individuals with neurological diseases View Publication

Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Pathogenic mutations in Bcl2-associated athanogene 3 (BAG3) cause genetic dilated cardiomyopathy (DCM),a disease characterized by ventricular dilation,systolic dysfunction,and fibrosis. Previous studies have demonstrated that BAG3 mediates sarcomeric protein turnover through chaperone-assisted selective autophagy to maintain sarcomere integrity in the human heart. Although mouse models provide valuable insights into whole-organism effects of BAG3 knockout or pathogenic variants,their utility is limited by species-specific differences in pathophysiology,which often do not translate to humans and contribute to the failure of clinical trials. As a result,the development of induced pluripotent stem cell-derived cardiomyocytes (iCM) and engineered heart tissues presents a promising alternative for studying adult-onset cardiac diseases. Here,we used genome engineering to generate an isogenic pseudo-wild-type control cell line from a patient-derived iPSC line carrying a pathogenic BAG3 variant,clinically presenting with DCM. While monolayer iCMs recapitulated some features of BAG3-mediated DCM,such as reduced autophagy,mitochondrial membrane potential,and decreased HSPB8 stability,they failed to develop the age-associated impairment in sarcomere integrity. Therefore,we developed a mature,patient-specific,human engineered heart tissue model of BAG3-mediated DCM and compared it to its isogenic healthy control. We demonstrated successful recapitulation of adult-like features of the clinically observed disorganized sarcomeres and impaired tissue contractility,thereby providing a platform to investigate BAG3-related pathophysiology and therapeutic interventions. Graphical abstract View Publication

The CBFA2T3::GLIS2 (CG) fusion protein causes aggressive pediatric acute megakaryoblastic leukemia (AMKL). Although dysregulated molecular pathways in AMKL have been identified,their role in early pre-leukemic transformation remains poorly understood. We developed a disease model utilizing genetically modified human induced pluripotent stem cells (hiPSC) physiologically and conditionally expressing CG. Using in vitro differentiation and single-cell multi-omics,we captured the impact of oncogene activity on gene-regulatory networks during hematopoiesis. We discovered that CG interferes with myelopoiesis through two alternative routes: by locking aberrant megakaryocyte progenitors (aMKP) in a proliferative state,or by impeding differentiation of aberrant megakaryocytes (aMK). Transcriptionally and functionally,aMKPs mimic CG-AMKL cells and establish a self-renewal network with co-factors GATA2,ERG,and DLX3. In contrast,aMKs partially sustain regulators of MK maturation but fail to complete differentiation due to repression of factors like NFE2,SPI1,GATA1 and LYL1. These insights may inform new strategies for targeting AMKL cell states. Subject terms: Acute myeloid leukaemia,Cancer models View Publication