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In vitro tissue models offer a flexible complementary study system for use alongside in vivo human tissue samples. Achieving accurate in vitro models relies on combining appropriate scaffolds, growth factors and cell populations to recreate human tissue complexity. Balancing a consistent cell supply with the creation of healthy tissue models can be challenging; established cell lines are often cancerous, with altered cellular function compared to healthy populations, and primary cells require repeated isolation, with associated batch-to-batch variation. Pluripotent stem cell-derived populations offer a consistent supply, as well as the ability to model disease phenotypes through cell reprogramming using patient-derived cells. In this study, we have used an induced pluripotent stem cell-derived fibroblast population to develop a dermal equivalent model. These cells form a consistent tissue construct with a structure and composition similar to primary fibroblast controls, which are able to support an overlying epidermis. The resultant full-thickness skin model demonstrates the expression of various key skin-related markers, correctly localised within the organised epidermis, notably improving on previous models of a similar nature. Providing proof of concept using an established in vitro protocol, this study paves the way for future work developing consistent, customised, full-thickness human skin equivalents using iPSC-derived populations.

Sex hormones, such as oestrogen and testosterone, display significant immune modulatory properties. This is highly relevant for transgender (trans) people who undergo gender-affirming hormone (GAH) treatment. However, only a limited number of studies have evaluated the immunological impact of GAH treatments, and almost none have assessed the impact in trans young people. Following recruitment to the Gender and IMmunity study (GIM) (n =100), biological samples were collected from trans young people (n = 47) including: trans males (birth-registered females taking testosterone-based GAH) and trans females (birth-registered males taking oestrogen-based GAH). All trans participants had taken GAH for at least 6 months. Samples were also collected from control individuals not taking GAH (n = 53). Immune profiles were evaluated using an 18-colour flow cytometry panel. In addition, the commercially available 37-parameter MaxPar panel was used for analysis of a subset of samples (n = 36) by mass cytometry (CyTOF). Immune cell abundance was compared across experimental groups, and correlated with plasma concentrations of oestradiol and testosterone using multiple regression models. From multiple comparisons analyses grouped by birth-registered sex, several differences were detected in the trans groups compared to control groups, in particular relating to abundance of B and T cell subsets. These differences appeared to be mainly associated with levels of plasma testosterone. The most notable differences were in trans males, who had lower numbers of CD11c+ B cells and higher numbers of CD4+ regulatory T cells (Tregs) compared to control females. Using CyTOF, further analysis of B and T cells subsets revealed the frequency of naïve B cells was higher in trans males compared to control females. This also correlated with testosterone concentration in this group. Differences in the abundance of other T cell subsets were detected in both trans males and trans females, however only a decrease in CD161+ T effector memory cells in trans males, compared to control females, was associated with lower testosterone levels. This cross-sectional observational study of young trans individuals suggests that testosterone treatment may have immune modulatory effects, which should be investigated further, including functional studies. While oestrogen treatment was associated with differences in some immune cells in trans females compared with controls, these were generally not associated with plasma oestradiol levels but rather with testosterone levels. Continued immunological research of young trans individuals taking GAH treatment is crucial for positive long-term health outcomes.

BackgroundProgressive photoreceptor loss in retinal degenerative diseases leads to irreversible vision impairment. Transplantation of human embryonic or induced pluripotent stem cell-derived photoreceptor precursor cells (PRPCs) offers potential for vision restoration. However, substantial early donor cell loss remains a major challenge. This study aims to elucidate the mechanisms underlying early PRPC loss and to evaluate host retinal responses to transplantation.MethodsPRPCs derived from human embryonic stem cells (hESC)-based retinal organoids were subretinally transplanted into both normal and degenerated canine retinas to investigate the impact of host retinal degeneration on transplant survival and integration. Single-cell RNA sequencing (scRNAseq) was performed on transplanted PRPCs 3 days post-transplantation into normal canine retinas, as well as on host retinal cells to identify molecular pathways associated with early donor cell loss. Non-invasive multimodal retinal imaging and immunohistochemical analyses were conducted to assess PRPC survival, integration, and host immune responses.ResultsDespite systemic immunosuppression, extensive early loss of human PRPCs occurred within the first week following xenotransplantation into both normal and degenerated canine retinas, suggesting that factors beyond immune activation contribute to donor cell loss. Transcriptomic analysis identified metabolic stress as a key driver of early donor cell death, characterized by dysregulation of mitochondrial function and oxidative phosphorylation pathways. Microglial infiltration into the donor cell mass was also observed in normal retinas, suggesting a response to donor cell stress and apoptosis. Beyond the initial phase of cell death, surviving donor cells integrated and persisted when transplanted into retinas with a partially preserved outer nuclear layer, whereas cell loss continued when intervention occurred at end-stage degeneration.ConclusionsMetabolic stress represents a critical barrier to PRPC survival following transplantation. Strategies aimed at enhancing metabolic resilience may improve transplantation outcomes. Furthermore, host retinal responses shape the transplant microenvironment, influencing donor cell survival and integration. These findings highlight the need for targeted interventions to mitigate early metabolic stress and optimize PRPC transplantation strategies for retinal degenerative diseases.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04509-w.

A major knowledge gap exists in understanding immune effects of adjuvants in early life. As environmental stimuli shape the infant immune system, adjuvants may also influence this process. Using a neonatal mouse model, we investigated the differential effects of adjuvants in neonates vs. adults. Mice were immunized with an adjuvanted hepatitis B vaccine followed by exposure to ovalbumin to determine whether prior immunization alters subsequent heterologous immune responses. Neonatal immunization with a Th2-biased alum-adjuvanted vaccine predisposed mice to develop Th2-biased immunity to subsequent ovalbumin exposures. Conversely, neonatal immunization with a Th1-polarizing CpG-adjuvanted vaccine resulted in preferential priming of Th1-biased heterologous responses. Immunization in adulthood did not alter heterologous immune responses. Early-life immunization modified the ability of bone marrow DCs to prime Th1/Th2 immune responses, suggesting a role for immune training in these antigen agnostic effects. These data suggest that rational adjuvant selection for early-life vaccines may beneficially shape immune development.

Both bacterial and viral infections can trigger an overwhelming host response, leading to immunopathology and organ dysfunction. Multiple studies have reported dysregulated myeloid cell states in patients with bacterial sepsis or severe SARS-CoV-2 infection. However, their relevance to viral infections other than COVID-19, the factors driving their induction, and their role in tissue injury remain poorly understood. Here, we performed a multi-cohort analysis of single cell and bulk transcriptomic data from 1845 patients across 25 studies. Our meta-analysis revealed a conserved severity-associated gene signature pointing to emergency myelopoiesis (EM) and increased IL1R2 expression in monocytes and neutrophils from patients with bacterial sepsis, COVID-19, and influenza. Analysis of tocilizumab-treated COVID-19 patients showed that IL-6 signaling blockade partially reduces this signature and results in a compensatory increase in G-CSF. To validate the role of these cytokines in vivo, we used a mouse model of influenza infection that recapitulates severity-associated increases in IL1R2+ monocytes and IL1R2hi neutrophils, and demonstrate that combined IL-6 and G-CSF blockade inhibits their production. Our study demonstrates the cooperative role of G-CSF and IL-6 in driving the production of severity-associated IL1R2+ myeloid cells and highlights the link between myeloid dysregulation and tissue injury during severe infection. Multi-cohort analysis of single cell and bulk transcriptional data combined with in vitro and in vivo experiments highlight the role of IL1R2+ myeloid cells on tissue injury during severe infection.

ABSTRACTProblemThe decidua is the interface between the uterus and the fetus. Studying decidual cells can reveal how healthy pregnancies are supported and mechanisms of pregnancy complications. There are two methods of obtaining decidual tissue following delivery. The placental bed can be suctioned following C‐section deliveries, or a thin layer of decidual tissue can be dissected from the placenta. This study aimed to compare immune cell populations obtained using the two methods.Method of StudyFrom individuals with scheduled C‐sections, we collected peripheral blood, decidua via vacuum suction of the placental bed, and decidua via dissection of the uterine‐facing side of the placenta. Samples were analyzed using a 22‐color full‐spectrum flow cytometry panel to identify immune cell subsets and functional markers.ResultsThe cellular composition of both decidual tissue collection methods were more similar to each other than to peripheral blood. Decidua collected via vacuum suction (Suc. decidua) had more live CD45+ cells. Decidua collected via dissection of the uterine‐facing side of the placenta (Plac. decidua) had significantly higher expression of Helios in CD4+ cells, suggesting more fetal T cells. Both types of decidual samples contained similar levels of Tr1‐like regulatory T lymphocytes expressing LAG3 and CD49b, whereas peripheral blood did not have this cell type.ConclusionCollecting decidual tissue using either method resulted in largely similar immune cell populations, suggesting studies are largely comparable regardless of whether samples were collected via suction or placental dissection. This will allow for greater flexibility in sample collection methods.

ABSTRACTErgothioneine (ET) is a naturally occurring thiol/thione that possesses several cytoprotective properties. Multiple studies suggest a potential neuroprotective role for ET. Here, we show in various Parkinson's disease (PD) models that ET is indeed neuroprotective. Firstly, using Drosophila genetic PD models, we demonstrated that ET treatment ameliorates the pathological phenotypes of parkin and LRRK2 PD mutant flies. This includes an improvement in their climbing score and the preservation of their dopaminergic neuronal number and mitochondrial integrity. Similarly, we observed the rescue of PD phenotypes by ET in mice treated with the Parkinsonian neurotoxin 6‐OHDA. This protective effect of ET is abolished in mice lacking OCTN1. Finally, we found that ET protects human LRRK2‐G2019S patient‐derived dopaminergic neurons from rotenone‐induced neurotoxicity; the action of ET is again OCTN1‐dependent. Collectively, our results strongly support a neuroprotective role for ET in PD and suggest that ET may be useful in the prevention and/or treatment of PD. We demonstrate here that the dietary compound Ergothioneine (ET) ameliorates the pathological phenotypes of a range of Parkinson's disease models, from Drosophila to mouse and patient‐derived human dopaminergic neurons. Our observations suggest that ET likely acts through the preservation of mitochondrial function to mediate its neuroprotective functions, and position ET as a safe and promising candidate to protect individuals against PD.

Establishment of the anterior-posterior (AP) axis is a critical symmetry-breaking event in mammalian development. In mice, this process involves the directed migration of the distal visceral endoderm (DVE). Here, we use targeted perturbations to demonstrate that asymmetric perforations in the basement membrane guide DVE migration. During implantation, matrix metalloproteinases in extra-embryonic tissues create uneven basement membrane perforations, establishing directional cues for cohesive DVE migration. Using light-sheet microscopy and tissue cartography, we show that migrating DVE deforms surrounding tissues. Physical modeling and live imaging of DVE protrusions indicate that basement membrane perforations orchestrate active force generation within the DVE. Extending these findings to human embryos and stem cell-derived models, we identify basement membranes with enriched perforations near the anterior hypoblast in embryos, suggesting a conserved mechanism for AP axis specification. These findings reveal an unrecognized role of basement membrane remodeling and mechanical heterogeneity in guiding directional tissue migration during mammalian development. Chen et al. show that in mice, extracellular matrix remodeling drives early migration of the anterior signaling center, establishing the body axis sooner than expected—a mechanism potentially conserved in humans.

SummaryPoly(ADP-ribose) polymerase 1 (PARP1) inhibition improved the ventricular function in Chagas disease (CD). Here, we uncovered that Parp1 depletion enhances cardiac health by regulating CD8+T cell response against Trypanosoma cruzi (Tc) infection. For this, Parp1−/− and wild-type (WT) mice were challenged with Tc and euthanized at acute and chronic phases of parasite replication and CD development, respectively. Parp1−/− mice controlled the chronic parasite persistence and associated inflammatory pathology more effectively than WT mice. Parp1−/− enhanced the maturation and stability of metabolically reprogrammed CD8+ effector and memory T cells with increased cytotoxic effects against the parasite. Mechanistically, PARP1 depletion enhanced the NFATc1 translocation to Pdcd1 promoter in CD8+T cells, altered the PD1:PDL1 stoichiometric ratio between CD8+T and antigen-presenting cells, and promoted CD8+T cell longevity and function during chronic Tc infection. We conclude that molecular and chemical inhibitors of PARP1 would offer a potential therapy to arrest CD pathogenesis. Graphical abstract Highlights•PARP1 inhibits NFATc1 binding to Pdcd1 promoter, impairing CD8+T cells maturity•PARP1 inhibition reprograms metabolic-immune profile of CD8+T cells in Tc infection•PARP1 inhibitors offer a potential therapy to control Tc infection and CD pathology Molecular biology; Immunology; Microbiology; Cell biology

The liquid biopsy (LB) represents a minimally invasive method for cancer screening that has been introduced in clinical practice for over a decade and that can accelerate treatment response assessment. LB allows the analysis of tumor cells or tumor-derived products (e.g. cell-free circulating nucleic acids, extracellular vesicles, and proteins) released from primary or metastatic tumor lesions into blood or other body fluids. In the era of immune-oncology, recent evidence indicates that tumor-specific immune responses can be detected in peripheral immune cells. The improvement of knowledge and the standardization of the isolation methods of these techniques will allow the detection and characterization of circulating tumor and immune biomarkers at an early stage as innovative tools to predict response to therapies. Nowadays, the analysis of peripheral blood mononuclear cells (PBMCs), circulating tumor cells (CTCs), peripheral blood-derived extracellular vesicles (EVs) and circulating tumor RNA (ctRNA) remains under-developed even if these non-invasive techniques can provide the complete genetic landscape of tumors and allow systematic tracking of cancer evolution. In addition, the evaluation of blood circulating cytokines, and early dynamics changes in the PBMCs of patients with solid tumors represent a promising area of research. Here, we present a comprehensive methodological framework for the evaluation of innovative peripheral blood-derived biomarkers. We also address the current challenges in isolation methods and analysis of PBMC, CTC, EVs and TEPs which are crucial for structuring the large amount of comprehensive information obtained from such samples, with the aim of advancing the translational cancer field. Graphical abstractImage 1 Highlights•Comprehensive detailed protocols for liquid biopsy applications.•Address the current challenges in isolation methods of circulating blood cells.•Reduces the gap due to the lack of standardized protocols.•Aims to advance the translational cancer field.

PurposeTo investigate whether individually targeting the outer mitochondrial membrane fission receptors FIS1 and MFF rather than the universally essential fission GTPase DRP1 is sufficient to suppress tumor initiating cells (TICs) without causing general mitochondrial dysfunction.MethodsFIS1 or MFF were silenced or knocked out in triple-negative breast cancer (TNBC) cells to investigate their essentiality for maintaining TICs in cell culture and xenograft models. We further investigate the impact of FIS1 deficiency on several functional properties of mitochondria including morphology, membrane potential and ROS production.ResultsWe demonstrate that FIS1 absence consistently suppressed TIC populations in cultured TNBC cells, and reduced tumor initiating activity in TNBC xenografts. Remarkably, we found that this phenotypic effect occurred in the absence of significant changes in ROS production, mitochondrial membrane potential and oxidative phosphorylation complex abundance even though FIS1-deficient TICs harbored a more reticular mitochondrial network. Finally, our in silico analyses established that all four DRP1 receptors (FIS1, MFF, MID49 and MID51) are ubiquitously expressed in healthy human tissues, and FIS1 is the most highly expressed DRP1 receptor in mammary gland.ConclusionOur data collectively suggest that FIS1 targeting should allow for the suppression of TICs in TNBC tumors without compromising mitochondrial functionality or causing major, systemic toxicity. We believe our findings have the potential to facilitate the development of TIC suppressing therapies for TNBC patients, which is of considerable clinical relevance given that this malignancy has very limited targeted treatment options and is associated with a high mortality rate.Clinical trial numberNot applicable.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12935-025-03909-5.

AbstractRNA modifications play a crucial role in various cellular functions. Here, we present ModiDeC, a deep-learning-based classifier able to identify and distinguish multiple RNA modifications (N6-methyladenosine, inosine, pseudouridine, 2′-O-methylguanosine, and N1-methyladenosine) using direct RNA sequencing. Alongside ModiDeC, we provide an extensive database of in vitro-transcribed and synthetic sequences generated with both the new RNA004 chemistry and the old RNA002 kit. We show that RNA modifications can be accurately recognized and distinguished across different sequence motifs using synthetic data as well as in HEK293T cells and human blood samples. ModiDeC comes with a graphical user interface and an Epi2ME pipeline that allows easy customization and adaptation to specific research questions, such as learning and classifying additional RNA modifications and further sequence motifs. The reproducibility across samples, together with the low rate of false positives, underscores the potential of ModiDeC as a powerful tool for advancing the analysis of the epitranscriptome and RNA modification. Graphical Abstract Graphical Abstract