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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.

Background: Crocodilians rarely develop cancer despite long lifespans and continuous exposure to environmental carcinogens, suggesting robust natural anti-tumor defense mechanisms. Methods: We investigated the anti-cancer activity of sera derived from the phylogenetically related species—alligators, crocodiles, and chickens, and studied their underlying immune mechanisms. The anti-tumor activity of alligator serum was tested in murine models of melanoma and lymphoma. Results: Alligator serum (AS) and its (NH4)2SO4-precipitated fraction (ASa) showed rapid and potent cytotoxicity toward multiple murine and human cancer cell lines while sparing non-malignant human cells. Importantly, ASa attenuated melanoma and lymphoma tumor growth in mice. Electrophysiological analyses in PN71 cancer cells treated with ASa revealed rapid membrane depolarization and formation of high-conductance pores consistent with Complement-mediated membrane attack complex (MAC) activity. Proteomic analyses identified the Complement component C5 as a major protein enriched in active fractions, implicating the Complement system in cancer cell killing. Based on phylogenetic similarity of C5, crocodile and chicken sera exhibit alligator-like comparable anti-cancer activity. Mechanistic studies in chicken serum showed that the anti-cancer activity depends on Ca2+ and Mg2+ ions, terminal Complement components (C5–C8), and IgM antibodies that initiate Complement activation. Immunodepletion of IgM from CSa significantly reduced cytotoxicity, whereas purified chicken IgM activated human Complement to induce cancer cell death. Conclusions: These findings identify a conserved IgM–Complement immune mechanism capable of selectively targeting malignant cells. The evolutionary conservation and cross-species functionality of this pathway highlight its potential as a bio-inspired strategy for developing novel Complement-based cancer immunotherapies.

Triple-negative breast cancer (TNBC) remains a significant challenge in oncology, contributing to a significant portion of cancer-related deaths among women. Current therapeutic options, including chemotherapy, surgery, radiation, and hormonal targeting therapies, exhibit limited efficacy, necessitating the exploration of innovative treatment modalities. The emergence of drug resistance and the persistence of cancer stem cells (CSCs) further emphasize the urgent need for novel therapeutic strategies. In this context, natural killer cell-derived extracellular vesicles (NK-EVs) have emerged as a promising cell-free therapeutic approach that exhibits high tumor infiltration and cytotoxicity against cancer cells and CSCs. This study aims to investigate the efficacy of NK-EVs as a therapeutic strategy for TNBC using various clinically relevant models, including patient-derived xenografts. Pathway analysis suggests strong activation of apoptosis via canonical caspase activation, as well as necrosis, thereby confirming the important cytotoxic effect of NK-EVs. Interestingly, NK-EVs were also found to suppress TNBC CSCs by disrupting their functionality and viability, and NK-EV treatment increased the expression of apoptosis markers in both CSCs and non-CSCs. By elucidating the therapeutic efficacy and translational potential of NK-EV-based interventions in TNBC, these findings offer critical insights for the development of future immunotherapeutic strategies against this aggressive subtype of breast cancer.

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.

Stem cell therapy has been widely studied as a promising treatment for spinal cord injury (SCI). However, a lack of functional scaffolds for stem cell therapy to address SCI leads to low therapeutic efficacy due to poor survival of transplanted cells. To address these challenges, this study aims to enhance regenerative potential of spinal cord organoids (SCOs) by employing extracellular matrix (ECM) recapitulating spinal cord-specific microenvironment. Decellularized spinal cord-derived ECM (ScEM) supports 3D culture for development, maturation, and functionality of human induced pluripotent stem cell-derived SCOs, comparable to standard organoid culture matrix such as Matrigel. Transplantation of SCOs using ScEM hydrogel promotes axonal regeneration with neovascularization in lesions, likely because of enhanced engraftment and integration of transplanted SCOs into defective tissues facilitated by ScEM. Accordingly, this approach induces early locomotor recovery of animals with SCI. These findings suggest that functional ECM scaffold capable of providing microenvironmental complexity of spinal cord can potentiate organoid-based therapeutics for SCI treatment. Graphical abstract Highlights•A decellularized spinal cord-derived ECM (ScEM) exhibits protein profiles resembling native spinal cord tissue.•Functional proteins related to neurodevelopment and regeneration are highly enriched in ScEM.•ScEM hydrogel supports development and maturation of spinal cord organoids (SCOs) comparable to Matrigel.•ScEM hydrogel enhances SCO-mediated axonal regrowth, immune modulation, and early recovery after spinal cord injury.

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro.

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However, methodologies for isolating functional synaptic structures from these models remain limited. Here, we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air‐liquid interface cerebral organoids (ALI‐COs) at distinct developmental stages (Day 90 and 150). Notably, the method avoids density gradients, requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g., GAP43) and classical synaptosomal proteins (e.g., PCLO, BSN, SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane‐enclosed GCPs with fibrous content and mitochondria in Day 90 isolates, and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl‐induced depolarization, which triggered phosphorylation changes in growth cone proteins (GAP43, MARCKS, MARCKSL1), cytoskeletal regulators (DCLK1, SHTN1, MARK4, MAP1B) and protein kinases (CAMK2G, PRKCE) in Day 90 GCPs, as well as classical synaptic vesicle cycle proteins (SYN1, DNM1, RPH3A) at Day 150. Overall, this study establishes a centrifugation‐based protocol for isolating growth cones and immature synapses from human organoids, capturing key stages of synaptic development and enabling scalable, patient‐compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders. Synapses are implicated in several neurological disorders and psychiatric diseases. The emergence and wide use of neural organoids provide a new opportunity to study human synapses in healthy and disease settings. Therefore, we developed a simple method for the enrichment of synaptosomes and growth cone particles from forebrain organoids. The method is based on differential centrifugation, works with small tissue amounts, and is highly reproducible. We validated the functionality of the isolated structures using KCl stimulation and phosphoproteomics. The method enables detailed mapping of protein composition and function during growth cone pathfinding, synaptogenesis, and establishment of neural circuits in organoids.

Background: Age-related macular degeneration (AMD) is a leading cause of vision loss. Reticular pseudodrusen (RPD), deposits on the apical side of the retinal pigment epithelium (RPE), signify a distinctive and critical AMD phenotype. Yet, their molecular basis and relationship to the conventional drusen seen in AMD remain unclear. Methods: We generated induced pluripotent stem cell-derived RPE cells from a clinically phenotyped cohort comprising only individuals with conventional drusen (AMD/RPD-) or with drusen coexisting with RPD (AMD/RPD +). To identify differences between the two cohorts, we performed single-cell transcriptomic, proteomic, quantitative trait locus (QTL) and transcriptome-wide association (TWAS) analyses, together with functional assays. Results: AMD/RPD + RPE cells exhibited enrichment of extracellular matrix (ECM) and hypoxia-responsive pathways, and a relative underrepresentation of mitochondrial and oxidative phosphorylation processes, when compared with AMD/RPD- cells. Genetic analyses supported shared modulation of mitochondrial pathways across AMD, with additional regulatory signals associated with RPD risk. Functionally, all RPE cohorts formed drusen-like deposits in vitro. AMD/RPD- lines generated more basal deposits, whereas AMD/RPD + cells exhibited increased susceptibility to monolayer disruption. Conclusions: These findings indicate that AMD with and without RPD represent mechanistically distinct entities and provide novel insight into the molecular mechanisms underlying disease heterogeneity in AMD.

Genome‐wide association studies (GWAS) have identified APOE2 allele as linked to exceptional longevity, with carriers exhibiting a reduced risk of Alzheimer's disease (AD). Apolipoprotein E (APOE), a glycoprotein involved in lipid transport, has three major alleles. However, alterations in lipid metabolism alone do not fully explain APOE2's protective effects. In contrast, APOE4 is the strongest genetic risk factor for AD. To investigate how APOE2 promotes neuronal longevity and confers neuroprotection, we generated human isogenic APOE iPSC‐derived models of both inhibitory GABAergic and excitatory neurons. In GABAergic neurons, APOE alleles differentially influenced endogenous DNA damage, DNA repair, and neuronal motility. Single‐cell RNA sequencing revealed APOE4‐specific gene expression signatures associated with AD, whereas APOE2 GABAergic neurons were enriched for DNA repair and signaling pathways. Consistent with this, APOE2 neurons exhibited significantly lower levels of DNA damage. APOE4 GABAergic neurons exhibit increased expression of repetitive ribosomal RNA, which is associated with DNA damage and cellular senescence. To determine whether the effects extended to excitatory neurons, we used a separate human model of Ngn2‐induced glutamatergic neurons, and found that APOE2 excitatory neurons were more resistant to cellular senescence and DNA damage than isogenic APOE3 and APOE4 neurons. Similarly, we found human APOE2‐targeted replacement mice exhibited less nucleolar enlargement and increased nuclear Lamin A/C, Hmgb1, and H3K9me3 compared to APOE4 counterparts. Together, our findings identify DNA repair and suppression of senescence‐associated processes as key mechanisms by which APOE2 is associated with neuronal resilience, providing mechanistic insight into its association with exceptional longevity and protection against AD. Neurons expressing APOE2 were more resistant to endogenous DNA damage, activated transcriptional signaling pathways associated with DNA repair, and were resilient to stress‐induced DNA damage and cellular senescence. In contrast, APOE4 neurons exhibited elevated expression of rRNA repetitive elements and were prone to becoming senescent.

Background: Diffuse Midline Glioma H3K27-altered (DMG) is an extremely aggressive and lethal childhood brain cancer that grows within the midline structure of the brain. Current treatment options are only palliative, making DMG in desperate need for therapeutic breakthroughs. One of the major challenges limiting the study of DMG is the lack of reliable preclinical models. In-vivo mouse models are expensive and technically challenging and in-vitro cell culture models lack the essential components of tumor microenvironment (TME) needed to recapitulate the complex biology of these tumors. Scalable human planar neural organoids (PNOs) with multi-cellular make-up can serve as a cost effective and reliable model system to capture DMG biology and allow effective species matched drug testing in-vitro. Methods: Using 3 separate DMG patient derived xenograft (PDX) cell lines, we spatially profiled a novel scalable human iPSC-derived PNO system containing neurons, functional astrocytes and microglia using the NanoString GeoMx spatial transcriptomics system. Results: We found that all three cell lines interact with and integrate into the human PNOs, demonstrating favorable growth conditions in a complex co-culture. Across spatially resolved regions of interest (ROI’s) tumor cells individually interact with microglia and astrocytes and transcriptomic profiling of these mixed cell ROI’s shows differences in the genetic signatures of both the normal cells (microglia/astrocytes) and the tumor cells. When compared to biopsies obtained directly from DMG patients, DMG cells within PNOs correlate strongly at both transcriptomic and proteomic levels. The multi-cellular PNOs also enabled drug target bystander toxicity screening not possible in a traditional tumor cell only monoculture. Conclusion: This study provides a proof-of-concept for scalable PNO modeling for DMG and underscores the translational relevance of this model system.

The interaction between human respiratory syncytial virus (HRSV) and the innate immune system has been demonstrated both in vitro and in vivo. Disruption of interferon (IFN) signaling pathways increases susceptibility and permissiveness to HRSV infection, whereas pretreatment of cells with IFN confers (partial) resistance. This suggests that HRSV disease severity is likely influenced by a pre-existing antiviral state of the respiratory epithelium, driven by baseline or primed expression of type I and type III IFNs. Here, we investigated whether prior exposure to respiratory bacteria or viruses alters in vitro susceptibility and permissiveness to HRSV infection by shaping an antiviral state using both immortalized cell lines and airway organoid models. In A549 cells, pre-exposure to S. aureus had the most significant impact by reducing HRSV infection and inducing robust interferon responses. However, this effect was not reproduced in airway organoids. Conversely, sequential virus infection experiments in airway organoids revealed that prior infection with human parainfluenza virus type 3 (HPIV‑3) reduced the spread of subsequent HRSV infection. In addition to interferon signaling this proved to be associated with epithelial damage mediated by HPIV-3 infection. Collectively, these findings show that HRSV susceptibility and permissiveness are influenced by the up- or downregulation of specific anti- and pro-viral factors induced by prior bacterial or viral exposure, together with the maintenance or disruption of epithelial integrity. Understanding these interactions could be crucial when identifying specific risk groups for severe HRSV-associated disease and the development of targeted HRSV interventions.

Diverse risk genes have been identified for neurodevelopmental disorders (NDDs), but how these genes converge on similar biological pathways in neurons, and thus give rise to similar phenotypes, is unclear. Here we apply a pooled CRISPR approach to successfully target 23 NDD loss-of-function genes with roles in chromatin biology and examine convergent effects on gene expression across human induced pluripotent stem cell-derived neural progenitor cells, glutamatergic neurons and GABAergic neurons. Points of convergence vary between these cell types, with the greatest number of convergent genes and strongest convergent networks in mature glutamatergic neurons, where they broadly represent synaptic, epigenetic and, unexpectedly, mitochondrial pathways. The most convergent networks were observed between NDD genes with shared biological annotations, clinical associations and co-expression patterns in human post-mortem brain. Drugs that were predicted to reverse convergent transcriptomic signatures and/or arousal and sensory processing behaviors ameliorated behavioral phenotypes in zebrafish NDD gene mutants. These results suggest that convergent effects of NDD risk genes could provide clinically useful insights. By studying 23 neurodevelopmental disorder genes across model systems and brain cell types, the authors uncovered shared downstream effects that converge on synaptic biology, epigenetic regulation and mitochondrial function.