Stem Cells Translational Medicine

AbstractThe lymphatic system is essential for maintaining fluid homeostasis, lipid transport and supporting immune function. Despite its central role in health and disease, advancements in understanding human lymphatic vasculature has been constrained, in part because primary human LECs are difficult to access and study in disease-relevant contexts. This study describes an efficient and scalable feeder-free method to differentiate human iPSCs into lymphatic endothelial cells (LECs) that are transcriptionally and phenotypically similar to primary fetal LECs. An iPSC-derived LEC system overcomes a drawback of primary cells by enabling precise genetic perturbations, supporting study of lymphatic diseases of interest in a human context. By grounding our approach in in vivo stages of lymphangiogenisis, we describe a staged protocol that recapitulates the key milestones of lymphatic development. We first adapted a published method to differentiate human iPSCs into venous endothelial cells (VECs) and then initiate transdifferentiation of VECs into LECs. Using immunocytochemistry, qPCR, as well as flow cytometry, we demonstrated expression of lymphatic-specific markers in the differentiated population. We further characterized our induced VECs (iVECs) and LECs (iLECs) through bulk RNA sequencing analysis and compared the populations to pseudobulk VEC and LEC transcriptomic datasets generated from human fetal heart endothelia at 12, 13 and 14 weeks of gestation. Through this work, we expanded the repertoire of approaches for accessing LECs, with the goal of accelerating discoveries in lymphatic biology and therapeutics. Abstract summary image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/712968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a9a406org.highwire.dtl.DTLVardef@4faec6org.highwire.dtl.DTLVardef@15b4e73org.highwire.dtl.DTLVardef@17b9c36_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.

Mast cells (MCs) are myeloid cells of the innate immune system. As a first line of defence they fulfill effector functions and immune modulatory properties. Upon activation they release pro-inflammatory mediators such as cytokines and proteases. It has been suggested that MCs may contribute to the development of liver fibrosis. However, investigating hepatic MC biology in mice is challenging due to low MC numbers and a lack of suitable detection techniques relying on MC proteins and their modifications. Here, we evaluated whether the expression strength of MC markers correlates with the degree of liver fibrosis in mice and aimed to determine the frequency and localization of hepatic MCs. We applied both a toxic (DEN/CCl4 treatment) and a genetic (Mdr2-/- mice) liver fibrosis model in C57BL/6 mice and found a significant correlation between fibrosis grade and the expression of several established mast cell markers. This correlation was further supported in patients with fibrosis and hepatocellular carcinoma (HCC) using publicly available transcriptomics datasets. We used FACS to purify and isolate MCs from fibrotic mouse livers and verified MC signatures by qPCR analysis of MC-specific gene expression. Hepatic MCs were predominantly negative for Mast-Cell-Protease 5 (Mcpt5) and occurred at a low frequency (approximately 1-2% of leukocytes). Using Molecular CartographyTM of fibrotic liver sections, we determined the spatial localization, expression signature, abundance (approximately 2 cells/mm2) and cellular environment of murine hepatic MCs. In summary, we demonstrated the existence of MCs in murine fibrotic livers and defined an MC expression signature that correlates with the strength of liver fibrosis. These findings will help to study MC biology in murine models of liver disease more effectively in the future.

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f059d9org.highwire.dtl.DTLVardef@9aeffforg.highwire.dtl.DTLVardef@27cd1org.highwire.dtl.DTLVardef@150b7d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells (HSCs) represents a promising approach to advance cell and gene therapy strategies yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by single cell RNA sequencing, which highlighted differentiation trajectories with preservation of lineage fidelity in committed progenitors. Defining a putative HSC cluster allowed an estimation of transduction efficiency in ex vivo cultures, which correlated with long-term gene marking in xenografts and patients enrolled in a gene therapy study. We then developed a clinically translatable, GMP-compliant process to expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult donors, by biologically informed protocol improvements of cytokine supplementation, media choice, timing of LV transduction and combinations of small molecules preventing the activation of differentiation programs. Our optimized process outperforms validated state-of-the-art cord blood expansion protocols when applied to mPB. LV integration site analysis and genomic barcode-based clonal tracking provided definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo culture. These results warrant clinical testing of this HSC transduction/expansion process in an upcoming clinical gene therapy trial for autosomal recessive osteopetrosis (EU CT 2024-518972-30). One Sentence SummaryA mobilized peripheral blood HSC expansion protocol optimized for gene therapy allows robust polyclonal long-term engraftment of LV-transduced cells.

Autologous cell suspension (ACS)-based therapies are an established strategy to enhance wound repair, yet limitations in preparation workflows and donor skin requirements remain barriers to wider clinical implementation. We have previously developed VeritaCell, a rapid enzymatic disaggregation-based approach that generates highly viable skin cell populations, including epidermal stem cell-enriched fractions, and demonstrated their pro-regenerative biological properties in vitro. Here, we have evaluated the in vivo efficacy of VeritaCell-derived ACS using a rat full-thickness excisional wound model. ACS preparations were applied at donor-to-wound area ratios of 1:1, 1:10, and 1:20, and wound progression was monitored through longitudinal image-based quantification alongside histological assessment of tissue architecture. ACS-treated wounds exhibited enhanced early wound closure dynamics, with significant within-group improvements evident by Day 6. Histological analysis demonstrated improved neo-epithelial organisation and reduced epidermal thickening in the 1:10 and 1:20 groups, with the 1:10 condition showing tissue architecture most closely resembling unwounded skin. Notably, beneficial effects were observed even at low estimated cell numbers, suggesting that cell viability and biological activity may be key determinants of therapeutic efficacy. Collectively, these findings provide in vivo validation of VeritaCell-derived ACS and support the use of biologically informed donor-to-wound coverage ratios. This approach may enable effective wound repair while minimising donor skin requirements, with potential relevance for the treatment of extensive injuries such as burns.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@11dfd34org.highwire.dtl.DTLVardef@1da6599org.highwire.dtl.DTLVardef@14427f0org.highwire.dtl.DTLVardef@19a242a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG

BACKGROUNDThe vascular endothelium is a dynamic tissue central to vascular homeostasis and disease, with endothelial cells (ECs) exhibiting plasticity that drives adaptive remodeling. Reelin, a secreted extracellular matrix glycoprotein critical for neuronal migration via ApoER2/VLDLR-DAB1 signaling, may also modulate vascular function and inflammation. However, its direct role in EC biology remains unclear. We investigated Reelin as a context-dependent signaling modulator in ECs, assessing its engagement of non-canonical pathways and regulation of endothelial plasticity relevant to cardiovascular pathology. METHODSHuman endothelial cells were stimulated with recombinant Reelin and analyzed by immunoblotting, immunofluorescence, and functional assays. Time-course studies assessed signaling, including phosphorylation of FAK, AKT, and DAB1 by Western blotting, while wound-healing assays quantified endothelial migratory capacity in vitro systems. RESULTSReelin rapidly robustly activated noncanonical signaling in endothelial cells, increasing FAK and AKT phosphorylation in a time-dependent manner consistent with cytoskeletal remodeling. Canonical DAB1 activation was limited. Functionally, Reelin enhanced migration, upregulated Endoglin/CD105, and induced a remodeling-associated phenotype. Reelin silencing altered endothelial phenotype, clearly indicating a role in homeostasis. Signaling was independent of VEGFR2 interaction. Overall, Reelin preferentially engages FAK/AKT pathways to drive partial phenotypic modulation without full endothelial-to-mesenchymal transition. CONCLUSIONWe show that Reelin is a previously unrecognized regulator of endothelial signaling and plasticity, acting via non-canonical FAK- and AKT-dependent pathways. By partially and dynamically modulating endothelial phenotype, Reelin promotes a remodeling-permissive state without triggering full mesenchymal transition. These findings identify Reelin as a novel modulator of endothelial function with potential implications for vascular remodeling and cardiovascular disease. What Are the Clinical Implications?Our findings identify Reelin as a modulator of endothelial signaling with a clear bias toward non-canonical FAK- and AKT-dependent pathways that regulate endothelial plasticity and remodeling. This signaling profile is highly relevant to vascular diseases in which endothelial dysfunction is driven by maladaptive cytoskeletal reorganization, altered migration, and persistent activation rather than complete loss of endothelial identity. The ability of Reelin to promote partial and dynamically regulated phenotypic modulation suggests that it may operate at early and potentially reversible stages of vascular pathology. In this context, dysregulated Reelin signaling could contribute to pathological vascular remodeling, including processes underlying atherosclerosis, fibrosis, and microvascular dysfunction. These results also raise the possibility that circulating or locally produced Reelin may serve as an indicator of endothelial activation state, providing a novel biomarker for vascular disease progression. Importantly, the identification of a signaling bias toward FAK- and AKT-dependent pathways highlights potential therapeutic targets downstream of Reelin that could be selectively modulated to limit maladaptive endothelial remodeling while preserving essential endothelial functions. Collectively, this study positions Reelin signaling as a previously unrecognized and potentially actionable pathway in the regulation of endothelial behavior, with direct implications for the development of targeted strategies aimed at preventing or attenuating cardiovascular disease progression

BackgroundActivated platelets release polyphosphate (polyP), a linear polymer of inorganic phosphate residues, from dense granules. Experiments performed under no-flow conditions show that polyP alters the kinetics of tissue factor (TF) pathway reactions, accelerating FXI activation by thrombin and FV activation by FXa and thrombin, and may impact inhibition by tissue factor pathway inhibitor (TFPI). How polyP influences this pathway in conjunction with platelet deposition under flow remains understudied. ObjectivesTo investigate how polyP-mediated acceleration of FV and FXI activation modulates thrombin generation under flow in TF-initiated coagulation. MethodsWe extended a previously validated mathematical model of platelet deposition and coagulation under flow to examine polyP-mediated effects following a small vascular injury during intravascular clotting. Simulations varied the surface density of TF exposed, wall shear rate, and plasma TFPI concentration. ResultsPolyP shifts the threshold TF density for a thrombin burst to lower TF densities. For TF densities above this threshold, polyP shortens the lag time to thrombin generation in a TF- and shear-rate-dependent manner. Although no explicit effect of polyP on TFPI function was included in the model, thrombin generation was much less sensitive to TFPI concentration with polyP, in a TF-dependent manner. Relative contributions of accelerations of FV and FXI activations depend on incompletely known enhancements by polyP. ConclusionsThe experimentally observed influence of polyP on TFPI function depends on TF density and may arise indirectly from accelerated FV and FXI activation, with the dominant effect arising through accelerated thrombin-mediated conversion of FV to FVa.

Background & AimsClonal hematopoiesis of indeterminate potential (CHIP) has been linked to chronic liver disease progression, yet its role across the full spectrum of metabolic dysfunction-associated steatotic liver disease (MASLD), from its initial development to end-stage complications, remains unclear. We aimed to comprehensively investigate the association of CHIP and its major subtypes with both the incidence and progression of MASLD. MethodsWe conducted a prospective cohort study of 353,218 UK Biobank participants, stratified into a healthy cohort free of MASLD at baseline (Cohort 1; n=230,270) and a prevalent MASLD cohort (Cohort 2; n=122,948). CHIP was ascertained from whole-exome sequencing data. We used multivariable Cox regression, competing risk models, and mediation analyses to assess the associations of CHIP (overall, by driver gene, and by clone size) with incident MASLD, cirrhosis, hepatocellular carcinoma (HCC), and liver-related death. ResultsIn Cohort 1, CHIP was associated with an increased risk of incident MASLD (HR 1.25, 95% CI 1.08-1.44) and cirrhosis (HR 1.57, 95% CI 1.10-2.25). These associations were driven by non-DNMT3A mutations, particularly TET2, and showed a linear dose-response relationship with clone size. In Cohort 2, non-DNMT3A CHIP was associated with progression to cirrhosis (HR 1.82, 95% CI 1.28-2.58). The associations were more pronounced in males and in individuals without obesity or diabetes. C-reactive protein partially mediated the CHIP-MASLD association. ConclusionCHIP, driven predominantly by non-DNMT3A mutations (particularly TET2) is an independent risk factor for both the development and progression of MASLD. These findings position CHIP as a novel player in the pathophysiology of MASLD and suggest potential avenues for risk stratification and targeted anti-inflammatory intervention. Impact and ImplicationsThis large-scale, prospective study establishes clonal hematopoiesis of indeterminate potential (CHIP) as a novel and independent risk factor for the entire spectrum of metabolic dysfunction-associated steatotic liver disease (MASLD), from its initial development to its progression to cirrhosis and liver-related death. For hepatologists and hematologists, these findings identify a genetically defined, high-risk subpopulation, particularly individuals with non-DNMT3A mutations, who may benefit from enhanced liver surveillance. The identification of systemic inflammation as a partial mediator of the CHIP-MASLD association suggests that anti-inflammatory therapies currently under development for liver disease could represent a targeted treatment strategy for this growing patient population.

BackgroundPrimary astrocyte cultures derived from neonatal rodent cortices provide a controlled system for investigating astrocyte-specific mechanisms. However, mixed glial preparations frequently contain contaminating microglia and oligodendrocyte progenitor cells, and most existing protocols require pooling tissue from multiple mouse pups to obtain sufficient astrocyte yields. This approach is impractical as it obscures sex and genotype, limits investigations of sex dependent astrocyte phenotypes, and precludes studies in certain transgenic models. To address this gap, our protocol achieves a high astrocyte yield from a single neonatal mouse brain, enabling sex- and genotype-specific cultures without the need for pooling. Mechanical removal of oligodendrocyte progenitors combined with pharmacological depletion of microglia using a Colony Stimulating Factor 1 Receptor (CSF1R) inhibitor produces highly enriched astrocytes suitable for functional assays, including those focused on sex-specific biology. MethodsCortical tissue was isolated from a single mouse pup is mechanically dissociated in astrocyte media. Cell suspensions are transferred to poly-D-lysine-coated flasks in astrocyte media. After 10-15 days in culture, OPCs are mechanically removed by horizontal shaking and microglia are selectively depleted by incubating cultures with CSF1R inhibitor PLX5622 for 24, 48, 72 and 96 hours. After PLX treatment, media is replaced and enriched astrocytes were maintained or passaged for experimentation. The sex of the pups is determined by PCR performed on DNA extracted from tail biopsies. ResultsImmunocytochemical analysis for astrocyte and microglia markers (GFAP and Iba1, respectively) showed that 24 hours of PLX5622 treatment did not fully eliminate microglia from mixed glial cultures. Extending treatment to 48 hours effectively depleted microglia while minimizing cytotoxicity and astrocyte loss and produced a pure, high-yield, sex-specific primary astrocyte culture. PCR reliably enabled the sex identification of pups used in culture using DNA extracted from tail biopsies. DiscussionThis protocol provides an efficient and reproducible method for generating high-purity, sex-specific primary astrocyte cultures from a single mouse brain. It improves consistency and purity while eliminating the need to pool tissue, preserving sex and genotype and enabling studies in transgenic mouse lines of both sexes.

Time-restricted feeding (TRF) is widely considered metabolically beneficial, yet its impact on chronic liver disease progression remains poorly defined. This study investigates the effects of TRF on liver fibrogenesis. Using carbon tetrachloride (CCl4)-induced, bile duct ligation (BDL)-induced, and choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced murine models of liver fibrosis, we demonstrate that TRF consistently exacerbates fibrotic injury. Mechanistically, TRF induces the systemic elevation of the ketone body {beta}-hydroxybutyrate (BHB). We identify the ketolytic enzyme 3-hydroxybutyrate dehydrogenase 1 (BDH1) as a critical mediator of this process within hepatic stellate cells (HSCs). BDH1 expression is markedly upregulated in activated HSCs, enabling these cells to metabolize BHB. This BDH1-dependent ketolysis redirects BHB-derived carbons into the tricarboxylic acid cycle, supplying acetyl-CoA and citrate to drive de novo lipogenesis and support a profibrogenic metabolic state. Both the genetic ablation of Bdh1 specifically in HSCs and the inhibition of hepatic ketogenesis successfully abolished the pro-fibrotic effects of TRF and exogenous BHB administration. Conversely, exogenous BHB alone was sufficient to recapitulate the exacerbated fibrotic phenotype observed with TRF. These findings reveal a context-dependent, detrimental role for TRF during chronic liver injury, driven by BDH1-mediated metabolic reprogramming in HSCs. Consequently, dietary interventions that elevate systemic ketone bodies should be approached with caution in the setting of active liver fibrosis.

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

MAIT are a highly versatile population of innate-like T cells that have been implicated in promoting tissue repair-associated process in a variety of tissue and diseases settings in the last years. While certain specific effector molecules responsible for MAIT-cell mediated have been identified, the mechanisms by which MAIT cells exert repair functions remain incompletely understood. Here, we show that hepatic MAIT cells express VEGFA, VEGFB and vimentin, an alternative ligand for the VEGFA-receptor VEGFR2 in both, regenerating and heathy tissue. Expression and secretion of these factors were induced in vitro by combined T cell receptor and cytokine stimulation. Supernatants of activated MAIT cells were able to promote proliferation of different epithelial and endothelial cells, including a liver sinusoidal endothelial-derived cell line in an VEGFR2-dependent manner. Together, our findings expand our understanding of MAIT cell function, especially in the liver and open new opens avenues for exploring MAIT therapeutic potential in modulating tissue repair.

Cirrhosis, the end stage of chronic liver disease marked by fibrosis and impaired liver function, is associated with cirrhosis-associated immune dysfunction, a condition in which systemic inflammation coexists with impaired host defense and increased susceptibility to infections. However, intestinal intraepithelial lymphocytes (IELs), key mediators of epithelial immune defense, remain poorly characterized in this context. Using high-dimensional profiling of paired duodenal biopsies and peripheral blood across disease stages, we define IEL alterations in cirrhosis. Contrary to prior reports of immune exhaustion, lymphocyte effector function was preserved, while disease progression was marked by systemic inflammatory remodeling and increased tumor necrosis factor (TNF) production by circulating T cells. The IEL compartment was markedly altered, with loss of CD8{beta} IELs, expansion of natural killer (NK) IELs, and reduced CCR9CD8{beta} IELs, suggesting altered gut homing. These findings refine cirrhosis-associated immune dysfunction as inflammatory immune reprogramming coupled to impaired epithelial immune surveillance. HighlightsPeripheral lymphocytes from cirrhosis patients retain effector capacity with enhanced inflammatory activity Cirrhosis reshapes the duodenal intraepithelial lymphocyte landscape Reduced frequency of CCR9+CD8{beta} IELs indicates altered gut-homing in cirrhosis

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

Intestinal organoids are three-dimensional in vitro structures derived from stem cells and serve as a valuable model for studying intestinal biology and pathophysiology. This study optimized the isolation, expansion, and differentiation of canine intestinal organoids from duodenum and colon. Organoids were generated from canine intestinal crypts and cultured in Matrigel with a growth factor cocktail. The impact of prostaglandin E2 (PGE2) concentration on organoid growth was evaluated, and a two-phase differentiation protocol--comprising patterning and differentiation media--was implemented, including interleukin (IL)-22 in the duodenal differentiation phase. Organoids cultured with 100 nM PGE2 exhibited increased crypt budding and organoid-forming efficiency, indicative of enhanced stem cell proliferation. Differentiated organoids expressed key intestinal markers (VIL1, SI, CHGA, MUC2), and forskolin-induced swelling demonstrated functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activity. Although the sample size (n=2) limits generalizability, this optimized protocol provides a relevant in vitro model for studying canine intestinal function. The model can be used in future research for disease modelling and translational applications, supporting downstream studies in gastrointestinal disease, drug permeability, and comparative One Health research.

BackgroundSex-related differences in cardiovascular disease suggest the presence of intrinsic vasoprotective mechanisms, with estrogen recognized as an important modulator of endothelial function. Building on existing evidence, the present study provides mechanistic insights into how estrogen and nitric oxide (NO) signaling regulate selective pathways of oxLDL uptake, mitochondrial dynamics, and inflammatory responses during early atherogenesis. MethodsWe combined an in vitro endothelial cell-macrophage co-culture model with in vivo studies in low-density lipoprotein receptor-knockout (LDLr-/-) mice to investigate the role of estrogen in early atherosclerotic processes. Human aortic endothelial cells (HAECs) were exposed to oxidized low-density lipoprotein (oxLDL) in the presence or absence of 17{beta}-estradiol (E2) and the nitric oxide (NO*) donor S-nitroso-N-acetylcysteine (SNAC). Key outcomes included oxLDL uptake, mitochondrial oxidative stress, mitochondrial dynamics, and inflammatory signaling. In vivo, male and female LDLr-/- mice were exposed to a short-term high-fat diet with or without SNAC treatment. Plasma lipid levels, blood pressure, aortic lesion formation, and cardiac remodeling were evaluated. ResultsE2 reduced oxLDL uptake and oxidative stress, effects recapitulated by SNAC; however, these responses involved distinct entry pathways, with E2 preferentially modulating lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) dependent uptake and SNAC targeting caveolae-associated mechanisms. In parallel, both E2 and SNAC reduced Scavenger Receptor Class B Type 1 (SR-B1) expression, suggesting an additional modulation on oxLDL transcytosis via this mechanism. Endothelial cells exposed to oxLDL exhibited altered mitochondrial regulatory proteins, including superoxide dismutase 2 (SOD-2), dynamin-related protein 1 (Drp-1), and optic atrophy protein 1 (OPA-1). Despite reducing oxidative stress, E2 increased the expression of adhesion molecules and enhanced monocyte adhesion in response to oxLDL exposure, particularly when combined with SNAC. Strikingly, E2 also modulated macrophage responses, increasing interleukin receptor antagonist (IL-1ra) expression and reducing GDF15, macrophage inhibitory factor (MIF), macrophage inflammatory protein 3 alfa (MIP-3), and matrix metalloproteinase 9 (MMP-9) levels, consistent with a less pro-inflammatory macrophage profile. In vivo, HFD increased plasma lipid levels and atherosclerotic lesion area in LDLr-/- mice, whereas SNAC partially attenuated these effects without affecting plasma lipid levels. In vivo, female LDLr-/- mice developed approximately 50% smaller aortic lesions than males, despite comparable or higher plasma lipid levels. A dyslipidemia led to increased blood pressure and a hypertensive phenotype in both males and females. SNAC treatment reduced lesion burden in both sexes and prevented diet-induced hypertension in females. ConclusionEstrogen limits early atherogenic injury by reducing endothelial uptake of oxLDL, preserving mitochondrial homeostasis, and modulating inflammatory signaling. Together, the E2 and NO pathways regulate early atherosclerosis through distinct yet complementary mechanisms, offering a potential framework for vascular-protective strategies.

BACKGROUNDInactivation of ANGPTL3 (angiopoietin-like protein 3, A3) is a proven therapeutic strategy for lowering plasma lipid levels independently of the LDL receptor (LDLR), yet the optimal approach to inactivate A3 remains unclear. A3 is proteolytically cleaved and circulates as full-length (A3-FL), N-terminal (A3-Nter) and C-terminal (A3-Cter) fragments. The specific contribution of each form of A3, and of its paralog, ANGPTL8 (A8), in modulating circulating levels of ApoB-Containing Lipoproteins (ABCLs) remain poorly defined. Clarifying these relationships will inform next-generation A3-directed therapies. METHODSWe performed liver perfusion studies to directly compare the number and composition of VLDL particles secreted from mice with and without A3. To amplify effects on cholesterol metabolism, we generated Ldlr-/- mice expressing wildtype A3 (A3-WT), A3-FL or A3-Nter, with or without co-expression of A8, and analyzed plasma lipids, circulating A3 and A8 complexes, and intravascular lipase activities. Complementary in vitro assays and structural modeling were used to assess relative endothelial lipase (EL) inhibition by A3 alone or in complex with A8. RESULTSLiver perfusion studies revealed that A3 inactivation does not alter the rates of hepatic secretion of VLDL in wildtype or Ldlr-/- mice. Inactivation of A8 alone lowered plasma LDL-cholesterol (C) levels by [~]20%, an effect dependent upon the expression of both EL and A3. Maximal inhibition of lipoprotein lipase (LPL) required co-expression of A8 plus both A3-FL and A3-Nter, indicating that A3 cleavage, in addition to A8 expression, is essential for maximal LPL inhibition. In contrast, A8 expression, but not A3 cleavage, was required for optimal EL inhibition. CONCLUSIONSA8 acts in concert with A3 to differentially modulate LPL- and EL-mediated lipolysis, which antagonizes hepatic clearance of newly-secreted atherogenic ABCLs. This mechanistic framework refines our understanding of A3-targeted lipid lowering and highlights the therapeutic potential of dual A3- plus A8-directed strategies to treat dyslipidemia and prevent atherosclerotic cardiovascular disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIInactivation of A3 lowers circulating ABCL levels without altering hepatic secretion rates of VLDL-ApoB or -TG. C_LIO_LIProteolytic cleavage of A3 is required for maximal inhibition of LPL. C_LIO_LIInactivation of A8 lowers LDL-C levels through an A3- and EL-dependent, but LDLR-independent, mechanism. C_LI What are the clinical implications?O_LICombining A8 inhibition with A3-inactivating therapies offers a strategy to achieve greater reduction in LDL-C levels and atherosclerotic cardiovascular risk. C_LI

Semaglutide has shown benefit in metabolic dysfunction-associated steatohepatitis (MASH), but real-world evidence across longitudinal liver phenotypes remains limited, particularly regarding how liver remodeling relates to weight loss and dose exposure. Using a de-identified federated electronic health record network spanning more than 29 million patients in the United States, including 489,785 semaglutide-treated adults, we analyzed 6,734 patients with baseline liver disease burden. We find that higher attained pre-landmark (0-2 years) semaglutide dose was associated with lower post-landmark (2-4 years) risk of steatohepatitis, alcoholic liver disease, and all-cause mortality, whereas greater pre-landmark weight loss was associated with lower post-landmark risk of steatohepatitis, steatotic liver disease, and hepatorenal syndrome, indicating distinct dose- and weight-linked patterns of long-term liver benefits. These associations were notable because semaglutide prescribing was generally lower during the post-landmark period, raising the possibility of durable benefit beyond peak exposure. Towards better understanding mechanistic bases for liver protection, we performed a complementary longitudinal study of 326 adults with paired noninvasive liver elastography measurements before and after treatment initiation. Median liver stiffness decreased from 4.85 [3.02 - 7.20] to 3.9 [2.6 - 5.8] kPa after semaglutide initiation (median change = -0.38 kPa; p<0.001), with 194 of 326 patients (59.5%) showing lower follow-up stiffness. A clinically meaningful reduction of at least 20% was observed in 133 of 326 patients (40.8%), and 69 of 326 (21.2%) shifted to a lower fibrosis stage by prespecified elastography thresholds. Larger improvements were also seen in patients with higher baseline stiffness (p<0.001); notably 80% of patients with cirrhosis-range baseline stiffness ([&ge;]12.5 kPa) achieved [&ge;]20% improvement versus 29.5% with minimal baseline disease (p <0.001). The proportion achieving at least 20% stiffness improvement was similar across weight-loss strata, including patients with no weight loss or weight gain and those with at least 10% weight loss (38.0% in each group), and liver stiffness change showed negligible correlation with changes in weight, BMI, HBA1c, alanine aminotransferase, or aspartate aminotransferase. To provide biological context, single cell RNA analyses demonstrated sparse overall hepatic GLP1R expression (0.0239%), with enrichment in non-parenchymal niches including cholangiocytes, intrahepatic cholangiocytes, liver sinusoidal endothelial cells, and hepatic stellate cells implicated in fibrogenesis and vascular remodeling. Together, this real-world evidence suggests diverse liver benefits for semaglutide beyond weight-loss with intricate dose response relationships.