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Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However, the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs), with unique design features, facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress, using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling, flow cytometry, and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blotting. Lipidomics provided insights into EV lipid composition, while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size, but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however, distinct differences were identified in lipid chain lengths and saturation levels, affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro. Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2–3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo, showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential, paving the way for future in vivo studies. The online version contains supplementary material available at 10.1186/s13287-025-04317-2.

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling, but its function in airway epithelial responses to IL-13, a key mediator in allergic diseases, remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing, providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes, including SPDEF, MUC5AC, POSTN, ALOX15, and CCL26, confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types, suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet, club, and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types, reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner, strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome, resulting in inconsistent findings across studies. Identifying a core set of genes consistently involved in COPD pathogenesis, independent of patient variability, is essential. We integrated lung tissue sequencing data from patients with COPD across two centers. We used weighted gene co-expression network analysis and machine learning to identify 13 potential pathogenic genes common to both centers. Additionally, a gene-based model was constructed to distinguish COPD at the molecular level and validated in independent cohorts. Gene expression in specific cell types was analyzed, and Mendelian randomization was used to confirm associations between candidate genes and lung function/COPD. Preliminary in vitro functional validation was performed on prioritized core candidate genes. Tissue inhibitor of metalloproteinase 4 (TIMP4) was identified as a key pathogenic gene and validated in COPD cohorts. Further analysis using single-cell sequencing from mice and patients with COPD revealed that TIMP4 is involved in ciliated cells. In primary human airway epithelial cells cultured at the air-liquid interface, TIMP4 overexpression reduced ciliated cell numbers. We developed a 13-gene model for distinguishing COPD at the molecular level and identified TIMP4 as a potential hub pathogenic gene. This finding provides insights into shared disease mechanisms and positions TIMP4 as a promising therapeutic target for further investigation. The online version contains supplementary material available at 10.1186/s12931-025-03238-1.

Development and lineage choice are driven by interconnected transcriptional, epigenetic and metabolic changes. Specific metabolites, such as α-ketoglutarate (αKG), function as signalling molecules affecting the activity of chromatin-modifying enzymes. However, how metabolism coordinates cell-state changes, especially in human pre-implantation development, remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring, characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover, increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather, it decreases acetyl-CoA availability, reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation, highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells, Embryology

SLC26A4 is the second most common cause of hereditary hearing loss worldwide. This gene predominantly harbors pathogenic variants, including splice, nonsense, and missense. Although missense variants are relatively common, their specific effects on protein function remain unclear. Consequently, there is an urgent need to establish an in vitro system to investigate how these variants impact SLC26A4 protein function. Genetic testing was conducted to determine the specific types of underlying genetic variants in patients. Following this, we employed plasmid transfection to evaluate the effects of the variants on both protein expression levels and the protein's subcellular localization. Thereafter, we transformed peripheral blood mononuclear cells (PBMCs) from the proband into induced pluripotent stem cells (iPSCs) through Sendai virus‐mediated transduction. Genetic testing revealed that the proband carried compound heterozygous variants: SLC26A4 c.919‐2A > G and c.317C > A. The c.317C > A variant markedly decreased the expression levels of SLC26A4 mRNA and its encoded protein. Additionally, it led to the protein's accumulation in the cytoplasm as aggregates. We successfully reprogrammed peripheral blood mononuclear cells from the proband into induced pluripotent stem cells (iPSCs) and verified that these iPSCs retained their pluripotency, differentiation potential, and genetic integrity. These results provide important insights into the mechanisms by which SLC26A4 gene variants lead to hearing loss.

Human brain development requires generating diverse cell types, a process explored by single-cell transcriptomics. Through parallel meta-analyses of the human cortex in development (seven datasets) and adulthood (16 datasets), we generated over 500 gene co-expression networks that can describe mechanisms of cortical development, centering on peak stages of neurogenesis. These meta-modules show dynamic cell subtype specificities throughout cortical development, with several developmental meta-modules displaying spatiotemporal expression patterns that allude to potential roles in cell fate specification. We validated the expression of these modules in primary human cortical tissues. These include meta-module 20, a module elevated in FEZF2 + deep layer neurons that includes TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical chimeroid experiments validated that both FEZF2 and TSHZ3 are required to drive module 20 activity and deep layer neuron specification but through distinct modalities. These studies demonstrate how meta-atlases can engender further mechanistic analyses of cortical fate specification. Subject terms: Developmental neurogenesis, Gene regulatory networks

Ceramides are bioactive sphingolipids that have physiological effects on inflammation, apoptosis, and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied, and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model, serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs), followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia), our data showed that following IR, there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis, such as CerS2 and CerS4 in CMs. Further, there was a considerable rise in the levels of total ceramide, long-chain (C16:0, C18:0, and C18:1), and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly, compared to CMs exposed to IR without inhibitor, our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels, reduced apoptosis, improved mitochondrial function, and enhanced survival of CMs exposed to IR. Furthermore, we used a transgenic mouse model, in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice, to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides, which causes increased apoptosis, proinflammatory cytokines, interstitial inflammatory cell infiltration, and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally, in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3.