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Progressive supranuclear palsy-Richardson’s syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however, the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here, we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation, neurofilament accumulation, and structural alterations of the axonal compartment, consistent with an early axonopathic phenotype. Notably, mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels, indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion, our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation, impaired structural homeostasis, and altered signaling pathways. Rather than acting as a primary driver, mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS.

Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases, but relevant human models are missing. Here, we present a robust 3D in vitro method with the use of human induced pluripotent stem cell–derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing, high-resolution immunofluorescence, and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells, pericytes, and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O2) for 1 week recapitulated key pathological features, including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases.

The potential of the immune system to decrease cancer progression is widely recognized and has led to the development of innovative anti-cancer immunotherapies. Here, we studied human macrophages derived from genetically engineered iPSCs (iMac) with angiotensin-converting enzyme (ACE) expression regulatable by a doxycycline (dox)-inducible promoter as a novel anti-cancer immunotherapy. Increased ACE expression in iMac (cells now termed ACE-iMac) augments polarization towards an M1 macrophage phenotype characterized by increased production of proinflammatory cytokines, reactive oxygen species, nitric oxide, and an RNA profile indicating an aggressive immune response. ACE-iMac kills tumor cells in vitro significantly better than iMac. In vivo, studies using tumor xenografts for melanoma, breast cancer, and head and neck squamous cell carcinoma (HNSCC) showed a highly significant 3.4- to 7.2-fold reduction in solid tumor size following ACE-expressing ACE-iMac immunotherapy as compared to results with iMac. To further investigate the impact of ACE on human anti-tumor responses, we developed a humanized BLT-NSG mouse model with a fully functional adaptive immune system. Here, ACE-iMac treatment significantly reduced the growth of human melanoma xenografts by enhancing the activation of human T cells and NK cells. In conclusion, enhancing ACE expression in human-derived macrophages (ACE-iMac) greatly amplifies their anti-cancer phenotype, offering a compelling new therapeutic strategy with the potential to improve clinical outcomes for cancer patients.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However, current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here, we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol, and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling, protein deposition, and epidermal differentiation. RDEB constructs recapitulated hallmark disease features, including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant, iPSC-derived skin constructs, offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine.

Gastrointestinal (GI) dysfunction, characterized by severe diarrhea and dehydration, is a central contributor to morbidity and mortality in filovirus disease in patients, yet the role of the epithelium in this clinical outcome remains poorly defined. Here, we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses, including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover, infection impaired adenylate cyclase signaling and CFTR-mediated ion transport, providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract, causing intense diarrhea and life-threatening dehydration. Yet, how these viruses affect the gut remains poorly understood. In this study, we used human mini-guts—small, three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues, disrupt key barrier structures, and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks.

Chemotherapy-induced peripheral neuropathy (CIPN) affects up to two-thirds of cancer patients undergoing cytotoxic chemotherapy. Here, we used human iPSC-derived sensory neurons (iPSC-DSN) to model CIPN in vitro. Administration of various chemotherapeutic agents (i.e., paclitaxel, vincristine, bortezomib and cisplatin) at clinically applicable concentrations resulted in reduced cell viability, axonal degeneration, electrophysiological dysfunction and increased levels of phosphorylated c-Jun in iPSC-DSN. Transcriptomic analyses revealed that the upregulation of c-Jun strongly correlated with the expression of genes of neuronal injury, apoptosis and inflammatory signatures. To test whether c-Jun plays a central role in the development of CIPN, we applied the small molecule inhibitor of the Jun N-terminal kinase, SP600125, to iPSC-DSN treated with neurotoxic chemotherapy. c-Jun inhibition prevented chemotherapy-induced neurotoxicity by preserving cell viability, axonal integrity and electrophysiological function of iPSC-DSN. These findings identify c-Jun as a key mediator of CIPN pathophysiology across multiple drug types and present preclinical evidence that c-Jun inhibition is an attractive therapeutic target to prevent CIPN.

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.

The differentiation of pluripotent stem cells into neurons is an essential area of biomedical research, with significant implications for understanding neural development and treating neurological diseases. This study compares neural cultures derived from a common induced pluripotent stem cell line (KYOU-DXR0109B) generated by two widely adopted methods: DUAL SMAD inhibition and exogenous NGN2 overexpression. The DUAL SMAD inhibition method, which differentiates through the neural stem cell stage, produces heterogeneous cultures containing a mix of neurons, neural precursors, and glial cells. Conversely, NGN2 overexpression generates more homogeneous cultures composed predominantly of mature neurons. Transcriptomic analysis revealed significant differences in neural gene markers expression profiles, with cultures from the DUAL SMAD inhibition method enriched in neural stem cell and glial markers, while NGN2 overexpression cultures showed elevated markers for cholinergic and peripheral sensory neurons. This study underscores the importance of choosing appropriate differentiation protocols based on the desired cell types, as each method yields neural cultures with distinct cellular compositions. Understanding these differences can help optimize protocols for specific research and therapeutic applications.

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.

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However, access to such cells is limited, and studies systematically mapping the spectrum of their inflammatory states are scarce. Here, we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust, accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling, revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli, including pro-inflammatory cytokines (TNFα, interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1), TLR3 (Poly(I:C)), TLR4 (lipopolysaccharide, LPS), and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g., oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally, the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1, LPS, or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism, highlighting the role of soluble mediators in the signal propagation. Altogether, this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation.