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ABSTRACTNatural clearance of hepatitis C virus (HCV) infection occurs in about 25% of primary infections, but offers only partial protective immunity against re‐infections. This study hypothesised that long‐lived polyfunctional HCV‐specific CD8+ memory stem T cells (TSCM) contribute to protective immunity in rare super‐clearer subjects who repeatedly clear viraemia. Six super‐clearers and four clearer‐chronic subjects who resolved a primary infection but subsequently developed chronic infection were studied at multiple timepoints. The TSCM population (CCR7+CD45RA+CD95+) was bulk sorted, labelled with CellTrace Violet (CTV), and stimulated in vitro for five days with cognate HCV peptide, IL‐2/IL‐15, and autologous PBMCs. Functionality of the expanded HCV‐specific TSCM was assessed via the proliferation, multi‐potency, and stemness indices. Total HCV‐specific CD8+ T cells from super‐clearers exhibited enhanced proliferative recall capability compared with clearer‐chronics. Furthermore, super‐clearers exhibited higher HCV‐TSCM frequencies post‐expansion (22.35 ± 34.35 vs. 2.41 ± 9.83; p = 0.0066). Notably, HCV‐TSCM in clearer‐chronics had ‘stemness’ indices of zero in samples before the re‐infection (i.e., no ability to generate TSCM as progeny), whereas super‐clearers consistently retained this key functional property. These findings suggest that the maintenance of self‐renewing HCV‐specific TSCM may underpin long‐term protective immunity against re‐infection and could inform vaccine design strategies targeting durable cellular memory. HCV re‐infection is common after spontaneous clearance of the initial infection. In the rare group of super‐clearers who clear repeatedly, HCV‐specific CD8+ T memory stem cells (TSCM) retain stemness, proliferation, and multipotency, sustaining immune protection. In contrast, clearer chronics lose these properties. TSCM self‐renewal underpins durable immunity and should inform vaccine design.

ABSTRACTThe stimulator of interferon genes (STING) pathway plays a pivotal role in innate immunity, acting as a key sensor of cytosolic DNA to initiate type‐I Interferon (IFN) and pro‐inflammatory cytokine production. This pathway is essential for host defence against bacterial, viral and other pathogenic threats and has emerged as a promising therapeutic target in cancer immunotherapy. However, conventional techniques such as immunoblotting and qPCR are limited in their capacity to study STING pathway activation in complex and heterogeneous biological systems, such as tumour masses or large cell populations. Here, we describe the application of mass cytometry (CyTOF) as a cutting‐edge approach to characterize the STING pathway at the sub‐population level. Using a high‐dimensional panel of metal‐labelled antibodies targeting key STING signalling components, we achieved resolution of pathway activation across diverse immune cell populations. This approach promises novel insights into cellular heterogeneity, pathway dynamics and the interplay between STING signalling and other immune pathways and underscores the power of high‐dimensional analysis to overcome the limitations of traditional methods to enable a more comprehensive exploration of immune signalling pathways. Using mass cytometry, we map the heterogeneous activation of the STING pathway at single‐cell resolution in cell lines and primary human immune cells and validate them with conventional techniques.

BackgroundThe immune system plays a pivotal role in progressing an injury through the healing cascade. However, comorbidities often lead to dysregulation of this response and are implicated in wound chronicity or stalling in the inflammatory phase, necessitating clinical intervention. The maternal-fetal interface, one of the most striking immunomodulatory microenvironments to be found in mammals, may be leveraged therapeutically in wound healing through the application of amniotic tissue allografts.MethodsThis study investigates the influence of dehydrated human amnion chorion membrane (DHACM) and lyophilized human amnion and chorion membrane (LHACM) on the inflammatory response of monocytes and macrophages in vitro. Human THP-1 monocytes and macrophages were challenged with lipopolysaccharide (LPS) or LPS + interferon gamma (INFγ), respectively, to model inflammatory conditions.ResultsLHACM and DHACM treatment significantly dampened inflammasome activity and pro-inflammatory protein production while enhancing cell survival in LPS-challenged monocytes. LPS/INFγ-challenged macrophages exhibited a phenotypic shift with treatment, synonymous with repair or regeneration functionality. This was further confirmed when these cells demonstrated corresponding attenuation of pro-inflammatory cytokine production, dampened inflammasome activity, and increased survival. Additionally, the rate of efferocytosis by DHACM and LHACM-treated macrophages was substantially elevated, indicating more efficient clearance of dead cell debris.ConclusionThese results indicate that DHACM and LHACM modulate the pro-inflammatory response of monocytes and macrophages, while enhancing the pro-reparative functions including efferocytotic capacity and cell survival. These data are suggestive of a potential cellular mechanism by which DHACM and LHACM may facilitate an efficient and appropriate inflammatory response to support the progression through the healing cascade.

Human stem cell-based embryo models provide experimentally amenable in vitro systems for developmental research. A key feature of embryo models is their multi-lineage differentiation, which allows for the study of tissue co-development. Here we develop human trunk-like structures that have morphologically organized somites and a neural tube that form through self-organized, endogenous signalling. Transcriptomic comparison with human embryo datasets suggests that human trunk-like structure cells approximate Carnegie stage 13–14 (28–35 days after fertilization). The absence of a notochord leads to a dorsal identity, but exogenous Sonic Hedgehog signalling activation ventralizes both the somites and the neural tube in a dose-dependent manner. We further identify reciprocal signalling: neural tube-derived cues induce medial ALDH1A2 in somites, which in turn generate retinoic acid signals that drive spontaneous neural-tube patterning. Together, our data highlight the value of modularity in embryo models, which we leverage to explore human trunk co-development. Makwana, Tilley et al. generate human stem cell-based trunk-like structures approximating Carnegie stage 13–14 of development. They use them to model and study the development of the thoracic and lumbar trunk.

BackgroundWNT signaling plays a key role in maintaining the gastric epithelium and promoting tumorigenesis. However, how gastric tumors achieve WNT niche independence remains unclear, as mutations on APC or CTNNB1—common mechanisms of ligand-independent WNT activation in colorectal cancer—are infrequent in gastric cancer. Understanding how WNT self-sufficiency is acquired in the stomach is therefore critical.MethodsWe analyzed mouse gastric organoids harboring oncogenic KRASG12D with or without RNF43/ZNRF3 (RZ) or CDH1/TP53 (CP) mutations, along with corresponding in vivo mouse models. Niche independence was assessed through growth factor withdrawal, Porcupine and pathway-specific inhibitor treatments, and WNT rescue assays. We performed single-nucleus multiome sequencing (RNA + ATAC) to investigate transcriptional and chromatin dynamics. Findings from mouse models were validated using patient-derived gastric cancer organoids, and pan-cancer cell line datasets were analyzed to evaluate clinical and cross-tissue relevance.ResultsGastric fibroblasts secreted canonical WNT2B to maintain the homeostatic gastric epithelium. Upon KRAS activation, epithelial cells were reprogrammed to secrete WNT ligands independently of additional mutations. Single-nucleus multiome analysis revealed that KRAS-driven MAPK signaling opened SMAD2/3-bound enhancers at the WNT7B locus, leading to the emergence of WNT7B-expressing subpopulations. Inhibition of SMAD2/3 phosphorylation suppressed both organoid growth and WNT7B transcription, whereas exogenous WNT restored organoid proliferation. Patient-derived organoids with HER2 amplification, KRAS amplification, or WNT2 copy-number gain exhibited Porcupine inhibitor-sensitive growth, indicating dependence on WNT secretion from the organoids. Analysis of public transcriptomic datasets further demonstrated that the KRAS–MAPK–WNT7B axis is conserved across other cancer types, including lung cancer.ConclusionsGastric tumors can bypass niche dependence by acquiring KRAS–MAPK–SMAD2/3-driven epithelial WNT secretion. Targeting this axis—through MAPK inhibition, SMAD2/3 blockade, or suppression of WNT secretion—may represent a therapeutic vulnerability in gastric cancer and other KRAS-high malignancies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02543-z.

Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet, new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here, we introduce PK Mito Deep Red (PKMDR), a low-phototoxicity mitochondrial probe for time-lapse imaging, whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψm). The positively charged PKMDR accumulates within mitochondria under a higher Δψm, leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψm, PKMDR’s fluorescence lifetime effectively reports on the status of oxidative phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM), we visualize heterogeneous Δψm across individual cells, organoids, and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall, PKMDR-FLIM is a robust tool for directly visualizing Δψm with high spatiotemporal resolution. Saimi and colleagues present PKMDR, a mitochondrial probe whose fluorescence lifetime serves as an indicator to monitor mitochondrial membrane potential and respiration dynamics with high resolution across cells and tissues.

Background: Infection with Mycobacterium avium paratuberculosis (MAP) is closely associated with Crohn's disease (CD) development, where excessive inflammation and marked intestinal damage are observed. Objectives: In this study, the role of short chain fatty acids, including propionic acid (PPA) and butyric acid (BA), was evaluated in an in vitro model, mimicking CD characteristics. Methods: MAP-infected THP-1 macrophages were treated with 1 mM and 10 mM of PPA or BA, and the conditioned media was co-cultured in Caco-2 cells. Results: Both PPA and BA caused an M2 shift with significant downregulation (p-value < 0.0001) in pro-inflammatory markers at both the RNA and protein levels. The downregulation is most likely due to the antimicrobial properties of PPA and BA. MAP growth was inhibited by several folds in MGIT (Mycobacteria Growth Indicator Tube) culture media supplemented with PPA or BA. Dysfunctional Caco-2 intestinal epithelial cells' integrity and function, due to MAP infection, were restored with PPA and BA treatment. Specifically, NOX1 expression was significantly decreased in 10 mM of PPA or BA-treated cells (p < 0.001), as validated by RT-PCR and microscopy. PPA and BA restored tight junction integrity by decreasing Claudin-2 expression in the MAP group. Conclusions: The data clearly demonstrated that short chain fatty acids contain anti-inflammatory and antimicrobial properties with downstream beneficial effects on damaged intestinal epithelial cells, suggesting potential benefits as a dietary supplement for CD patients, particularly those who are not pregnant, due to a possible increased risk of autism spectrum disorder (ASD) development in offspring associated with propionic acid exposure.

Ovine colostrum exosomes obtained from nutritionally programmed dairy ewes (F0) may present modifications in microRNAs, thus having consequences for the intestinal barrier function and immunity parameters of lambs (F1). To test this hypothesis, colostrum exosomes from two ewe groups [F0-MET (nutritionally programmed ewes being fed methionine during early life) and F0-CTRL (ewes not supplemented with methionine during early life)] were sequenced to compare differences in the miRNAome. In addition, these exosomes were added to an in vitro co-culture in a Transwell chamber system consisting of ovine duodenum intestinal organoids and macrophages to assess the expression of genes encoding tight junction proteins in organoids and immunity parameters in macrophages. Finally, the concentrations of cytokines (e.g., IL-12 and IL-6) were assessed by ELISA kits in the supernatants of the chamber containing macrophages. According to the miRNAome, the expression of two miRNAs (e.g., oar_miR_376c_3p and oar_miR_432) was reduced in the colostrum exosomes obtained from dairy ewes nutritionally programmed with dietary supplementation of methionine during early life (F0-MET ewes). These changes did not seem to modify the expression of intestinal barrier and immune response marker genes when these exosomes were added to a co-culture of ovine intestinal organoids and macrophages. However, the levels of IL-12 produced by macrophages were reduced (p < 0.05), which suggests the inhibition of inflammatory pathways. Further studies using ovine colostrum exosomes obtained from nutritionally programmed ewes will help to clarify their potential to improve the health of suckling lambs.

Pathogenic GRN variants that reduce progranulin (PGRN) levels cause frontotemporal dementia (FTD). To facilitate model development, we generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of two family members carrying the GRN c.1009C>T (p.Q337X) pathogenic variant—one symptomatic and one asymptomatic—as well as a non-carrier first-degree relative serving as a genetically matched control. The obtained iPSC lines were validated for pluripotency markers (Nanog, Sox2, Oct4, and TRA1-1-81), genomic integrity, and differentiation potential. The obtained iPSC lines were subsequently directed toward neuroepithelial stem (NES) cells. NES identity was confirmed by the expression of lineage-specific markers, including Nestin and Sox2 (assessed by immunocytochemistry), as well as SOX1, PLAGL1, and MKI67 (evaluated by real-time PCR). Furthermore, GRN mRNA levels were significantly reduced in iPSC and NES lines derived from mutation carriers compared to control cells. The established iPSC and NES cell lines represent a platform for modeling progranulin-deficient FTD. The symptomatic and asymptomatic carrier-derived lines obtained from the same family offer a unique opportunity to study disease progression across clinical phases. The control line, derived from a related (first-degree) non-carrier, minimizes genetic background variability. Their utility of the established cell lines extends to therapeutic drug screening and further differentiation into neuronal, non-neuronal, and organoid models.

Single-cell profiling technologies allow exploring molecular mechanisms that drive development, health, and disease. However, current methods still fall short of profiling single cell transcriptomes comprehensively, with one major challenge being high non-detection rates of specific transcripts and transcript regions. Such information is often crucial to understanding the biology of cells. Here, we introduce RoCK and ROI (Robust Capture of Key transcripts and Regions Of Interest), a scRNA-seq workflow encompassing two techniques. RoCKseq uses targeted capture to enrich for key transcripts, thereby supporting the detection and identification of cell types and complex phenotypes in scRNA-seq experiments. ROIseq directs a subset of reads to a specific region of interest via selective priming. Importantly, RoCK and ROI enables retrieval of specific sequence information without compromising overall single cell transcriptome information. We validate RoCK and ROI across diverse biological systems highlighting the versatility and showing the power of the method to retrieve critical transcriptomic features. Current single-cell RNA sequencing methods struggle to comprehensively profile transcriptomes, with many lowly expressed transcripts remaining undetected. Here authors present a workflow for enhancing the detection of both transcripts and regions of interest in combination with a standard transcriptome profile.

Ulcerative colitis (UC) is associated with epithelial metabolic derangements which exacerbate gut inflammation. Here, we develop colon organoid (colonoid) lines from pediatric patients with endoscopically active UC, inactive UC, and those without intestinal inflammation to interrogate functional metabolic differences in the colon epithelia. We demonstrate that active UC colonoids exhibit hypermetabolic features and cellular stress, specifically during differentiation. Hypermetabolism in active UC colonoids is driven, in part, by increased proton leak, and excess lipid accumulation. Active UC colonoids exhibit heightened activation of the master lipid regulator PPAR-α and its transcriptional pathways. Pharmacological PPAR-α inhibition limits lipid accumulation, induces a metabolic shift towards glucose utilization, suppresses hypermetabolism, and reduces chemokine secretion and cellular stress markers. Collectively, our findings identify lipid-related metabolic dysfunction as a key pathologic feature of the pediatric UC epithelium and highlight the potential of patient-derived colonoids as a preclinical model for evaluating epithelial-targeted therapies addressing this dysfunction. Ulcerative colitis (UC) is associated with epithelial metabolic derangements which exacerbate gut inflammation. Here the authors report that colonoids from children with ulcerative colitis exhibit hypermetabolism and cellular stress primarily driven by lipid dysregulation. Pharmacological inhibition of PPAR-a, a transcriptional regulator of lipid metabolism, alleviates epithelial stress and inflammation.

Self-assembly is a fundamental property of living matter that drives the three-dimensional organization of cell collectives such as tissues and organs. Here, the co-assembly of synthetic and natural cells is leveraged to create hybrid living 3D cancer cultures. We screen a range of synthetic cell models for their ability to form augmented tumoroids with artificial but controllable micro-environments, and show that the balance of inter- and extracellular adhesion and synthetic cell surface tension are key material properties driving integrated co-assembly. We demonstrate that synthetic cells based on droplet-supported lipid bilayers can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids and eliminating the need to integrate complex living immune cells. Using the ART-TIME approach, we identify a AhR-ARNT-mediated co-signaling mechanism between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our study advances the field of hybrid organoid engineering, offers opportunities for the construction and modelling of artificial tumour environments, and marks a step towards the design of functional living/non-living cytomimetic materials. Synthetic cells have huge potential in model systems. Here, the authors engineer synthetic–living hybrid tumoroids that replicate tumour-immune interactions in 3D, study synthetic cells integration, and demonstrate systematic studies of immune evasion and T cell engager therapies.