Stem Cells Translational Medicine

ObjectivesCardiac regenerative therapy using human induced pluripotent stem cell (hiPSC)-derived tissues and organoids holds great promise for treating heart diseases. Successful clinical translation requires biomimetic cardiac tissues that not only recapitulate native myocardial architecture but also actively integrate with host vasculature. We aimed to engineer self-organized, vascularized cardiac microtissues (VCMs) and evaluate their therapeutic and regenerative potential in a rat model of myocardial infarction (MI). MethodsVCMs composed of hiPSC-derived cardiomyocytes, vascular endothelial cells, and vascular mural cells were cultured under dynamic conditions to promote self-organization and prevascular network formation. One week after MI induction by coronary artery ligation in athymic immunodeficient rats, VCMs were transplanted onto the infarcted myocardium. Cardiac function was assessed by echocardiography and magnetic resonance imaging. Three-dimensional host-graft vascular architecture was visualized by light-sheet fluorescence microscopy following tissue clearing, and functional perfusion was evaluated by intravenous DyLight 488-conjugated lectin injection via host systemic circulation prior to tissue harvest. ResultsVCM transplantation significantly improved cardiac function and reduced infarct size compared with controls. Histological analyses demonstrated enhanced graft survival and neovascularization. Three-dimensional imaging revealed human-derived self-organized vascular networks within engrafted VCMs. Lectin perfusion confirmed functionally perfused, reciprocal host-graft vascular integration, including extension of graft-derived vessels into host myocardium, accompanied by myocardial regeneration. Early graft engraftment was significantly higher in the VCM group than in non-prevascularized controls. ConclusionsSelf-organized prevascularization of hiPSC-derived cardiac microtissues enable active host-graft vascular integration through functional vascular networks, thereby enhancing myocardial regeneration and therapeutic efficacy. This strategy represents an advanced approach for cardiac regenerative medicine. SummaryThis study aimed to develop self-organized, vascularized cardiac microtissues (VCMs) derived from human induced pluripotent stem cells (hiPSCs) and to evaluate their myocardial regenerative potential in a rat model of myocardial infarction (MI). VCMs were engineered from hiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells and cultured under dynamic conditions to enable self-organization and prevascular network formation. One week after MI induction, VCMs were transplanted onto the infarcted myocardium. Cardiac function was evaluated using echocardiography and magnetic resonance imaging. Light-sheet fluorescence microscopy combined with tissue clearing was used to visualize three-dimensional vascular architecture and host-graft integration, while lectin perfusion analysis assessed functional blood flow. VCM transplantation significantly improved cardiac function, increased early graft engraftment, and enhanced neovascularization. Importantly, self-organized human-derived vascular networks within the VCMs actively integrated with the host vasculature, forming functional, perfused host-graft vascular connections. These findings indicate that prevascularized VCMs do not merely survive after transplantation but actively promote vascular integration and myocardial regeneration through functional vascular networks. Together, these results demonstrate that self-organized vascularization markedly enhances graft integration, survival, and therapeutic efficacy, underscoring the clinical potential of VCM-based strategies for cardiac regenerative therapy.

BackgroundChronic limb-threatening ischemia (CLTI) is the most severe form of peripheral artery disease and can result in debilitating tissue damage, limb loss, and mortality if left untreated. Despite surgical bypass and endovascular interventions, there is high unmet need to develop novel therapies that can restore durable blood flow and rescue limb function in patients whose disease is not amenable to surgical bypass and endovascular procedures. Human induced pluripotent stem cell (hiPSC)-derived vascular progenitor cells (VPC) hold promise for addressing this unmet need, yet their clinical adoption will require a scalable and consistently high-quality cell product that can be used safely in a large number of CLTI patients. MethodsHere, we report a robust, scalable GMP-adaptable platform for generating universally immuno-compatible VPC from human leukocyte antigen (HLA) class I/II-edited hiPSCs with extensive characterization of phenotypic and functional attributes critical to address key translational gaps in developing cell-based therapies for CLTI. We have interrogated their therapeutic efficacy in multiple murine CLTI models using a combination of clinically relevant endpoints, histology, and tissue-based RNAseq analysis. ResultsWe found that VPC-treated mice exhibited significantly improved perfusion ratios and preserved limb function, reduced inflammation, and increased physiological neovascularization without pathological malformations. ConclusionsGenetic modification conferring hypoimmune status coupled with a robust differentiation process enables large scale production of an "off-the shelf" high-quality VPC product with the potential to address unmet need in CLTI patients regardless of HLA status.

Human induced pluripotent stem cell (hiPSC) technologies offer human-relevant cardiac models for biomedical applications. However, workflows for differentiation of cardiac stromal cells and fabrication of engineered heart tissue (EHT) commonly rely on animal serum, contrary to growing policy demands to reduce use of these products. Applying marker analysis via COL1A, DDR2 and GATA4 for cardiac fibroblasts or CD31, CD34 and CD144 for endothelial cells, we tailored Panexin, a defined serum substitute, to support high efficiency differentiation of cardiac stromal lineages to 85% purity without additional purification steps. We evaluated fabrication of EHTs using hiPSC-cardiomyocytes only (monoculture) or further combined with cardiac fibroblasts and endothelial cells (triculture; 70%:15%:15%, respectively). Panexin poorly supported fabrication and contractility of EHTs, a finding unaltered by modulating spontaneous cardiac myofibroblast activation via TGF{beta} inhibition. In contrast, human serum enabled fabrication of mono- and tri-culture EHTs, wherein constructs made without TGF{beta} signalling inhibition delivered the strongest contractile forces (up to 0.25 mN) and exceeded comparator tissues engineered using animal serum. Our data show that iterative evaluation of serum substitutes, human serum, cell combinations and signalling pathway modulators can mitigate use of animal serum for functional EHT generation, aligning with the UK governments roadmap for alternative methods.

The most severe phenotype of alpha-1-antitrypsin deficiency (AATD) is caused by the Z-mutation within the SERPINA1 gene. The Glu342Lys substitution causes misfolding and polymerisation of the alpha-1-antitrypsin (AAT) protein, its accumulation in the ER and increases the susceptibility of hepatocytes towards ER-stress. Here, we present an induced pluripotent stem cell (iPSC)-based hepatic model to study AATD. We demonstrate that iPSCs from AATD patients differentiate equally well to hepatocyte-like cells (HLCs) as control iPSCs. We detected ZAAT polymers in patient-derived HLCs which could be reduced by SAHA or CBZ treatment. Transcriptome analyses revealed major differences in metabolism and signalling between control and AATD HLCs and indicated increased stress levels affecting intracellular organelles. Importantly, the transcriptomes of control and patient-derived cells separated into distinct clusters with respect to the expression of Heat-shock protein (HSP) encoding genes. SAHA treatment increased expression of various HSPs which might contribute towards reduced ZAAT polymers.

Donor organ shortage remains the major barrier to transplantation resulting in deaths on the waiting list. For lungs, aspiration-related injury is a common cause of donor organ discard and increases the risk of primary graft dysfunction. Currently, no effective therapies exist to repair damaged donor lungs prior to transplantation. Here, we investigated whether mesenchymal stromal cells (MSCs) from bone marrow or full-term amniotic fluid could restore severely injured donor lungs in a porcine model integrating ex vivo lung perfusion, transplantation and post-transplant follow-up (n=48; 24 donors, 24 recipients). MSCs were administered either once during ex vivo lung perfusion or repeatedly across lung perfusion and the early post-transplant period and compared with placebo treated controls. A single dose conferred only partial benefit, whereas repeated dosing restored graft function, normalized gas exchange and haemodynamics, and prevented graft dysfunction. MSCs from both sources were similarly effective in repeated regimens. These findings identify dosing schedule, rather than cell source, as key determinant of durable organ rescue and support perfusion-guided cell therapy as potentially generalizable regenerative strategy across solid-organ transplantation.

BackgroundHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) constitute an attractive system for basic research and pharmacologic screening of new molecules of clinical interest. Numerous protocols aiming at differentiating atrial- or ventricular-like cardiomyocytes (hiPSC-CMs) are available. Conversely, only a few are available for obtaining patient-derived sinoatrial node-like pacemaker myocytes (PM-hiPSC-CMs). Here we validate a new protocol to differentiate mature PM-hiPSC-CMs as a model of native sinoatrial node (SAN) myocytes. MethodsWe generated PM-hiPSC-CMs through a 2D matrix-sandwich method promoting epithelial-to-mesenchymal transition and small molecule-based temporal modulation of Wnt signaling pathway. In addition, we treated our cells with triiodothyronine, dexamethasone and intracellular cyclic AMP (DTA) to enhance expression of proteins involved in intracellular Ca2+ handling. ResultsProteomic analyses showed expression of key SAN proteins in DTA-treated PM-hiPSC-CMs. Importantly, expression of proteins related to Ca2+ handling was increased in DTA-treated PM-hiPSC-CMs compared to untreated ones. DTA-treated PM-hiPSC-CMs displayed action potentials, ionic currents and intracellular Ca2+ dynamics typical of native SAN. In addition, pacemaker activity responded to both {beta}-adrenergic and muscarinic stimulation. ConclusionsOur data indicate that the differentiation protocol effectively generates PM-hiPSC-CMs with typical native human SAN features. This protocol may serve as a potential approach to generate PM-hiPSC-CMs from patients with history of sinoatrial node disfunction (SND) carrying different mutations in ion channels underlying pacemaking. In addition, these in vitro models of SND could be used for testing long-term vector-based gene therapeutic strategies to handle bradycardia.

Epigenetic modulators such as histone deacetylases (HDACs) and histone acetyltransferases (HATs) are known master regulators of gene expression that substantially impact cardiac electrophysiology. Novel pharmacological agents, HDAC inhibitors, are rapidly emerging as treatments for cancer and immune diseases, and their effects on cardiac ion channels (ICs) are of great interest. We used small interfering RNAs to individually suppress each of the known HDACs, including sirtuins (SIRTs), in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), iCell2. Follow-up deep-sequencing allowed comparison to identically processed and normalized RNA sequencing data from adult human left ventricle (LV) from the GTEx database. The transcriptomics analysis revealed high similarity of gene expression patterns for cardiac ICs (with some differences in calcium influx and calcium buffering related genes), as well as strong co-regulation by cardiac transcription factors (TFs) and HDACs/SIRTs in both hiPSC-CMs and the adult LV. Partial least square regression models helped visualize links between HDACs/HATs, TFs, and cardiac ICs and helped identify potential key regulators of cardiac IC transcription. Powerful TFs, including MEF2A, GATA4, 6 exerted positive effect on IC genes while RUNX1 and SHMT2 were distinct negative regulators in both sample types; TRIM28 was found to serve opposite roles in the two sample types. In functional measurements, HDAC suppression primarily increased excitability, while SIRT suppression decreased excitability, in line with transcriptomic links. Our analysis offers insights about the role of epigenetic modifiers in regulating cardiac electrophysiology and informs the utility of hiPSC-CM as a scalable experimental model for cardiotoxicity testing of HDAC inhibitors.

BackgroundCurrent heart failure (HF) biomarkers (e.g., BNP/NT-proBNP) have limitations in specificity and performance in HF with preserved ejection fraction (HFpEF). Circulating microRNAs (miRNAs) are promising novel biomarkers. This study aimed to comprehensively evaluate the diagnostic stability of circulating miRNAs for HF, identify novel candidates, and prioritize them for clinical translation. MethodsWe conducted a systematic review and meta-analysis. PubMed, Embase, and Cochrane Central were searched from inception to March 2025. Studies comparing miRNA expression in HF versus control groups using blood or tissue samples were included. Data were extracted, and study quality was assessed using the Newcastle-Ottawa Scale (human) and SYRCLEs tool (animal). A random-effects model pooled log odds ratios (logORs) for each miRNA. Subgroup analyses were based on species, ethnicity, and sample type. Evidence quality was graded using the GRADE framework. ResultsEighty-six studies (61 human, 25 animal) with 3,023 samples were included. Meta-analysis identified 71 consistently dysregulated circulating miRNAs (58 up, 13 down) in HF. Key upregulated miRNAs included miR-21 (logOR=8.15, 95% CI: 7.55-8.74), miR-423-5p, and miR-210. Key downregulated miRNAs included miR-144 and miR-126. Subgroup analyses revealed differences by species, ethnicity (Asian vs. non-Asian), and sample type (serum vs. plasma). GRADE assessment classified five miRNAs (miR-1, miR-21, miR-221, miR-423-5p, miR-148a) as high-quality evidence. ConclusionsThis meta-analysis identifies a panel of circulating miRNAs with stable expression in HF, with miR-21 and miR-423-5p being the most robust. Evidence grading provides a clear priority list (e.g., miR-21, miR-423-5p) for clinical validation. Subgroup heterogeneity highlights the need for standardized protocols and precision diagnostics in future biomarker development.

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

BackgroundAnthracyclines such as doxorubicin are effective chemotherapeutics but are limited by cardiotoxicity driven in part by mitochondrial dysfunction. Dysregulated mitochondrial dynamics, particularly excessive dynamin-related protein-1 (Drp1)-mediated fission, contribute to doxorubicin-induced cardiac injury and support selective survival of cancer cells. ObjectivesTo determine whether DRP1i2, a novel small molecule Drp1 inhibitor targeting a conserved domain shared between human and mouse, can function as a cardio-oncology therapeutic by reducing doxorubicin-induced cardiotoxicity while maintaining or enhancing anti-cancer efficacy. MethodsCardioprotective effects of DRP1i2 were evaluated in a murine model of chronic doxorubicin cardiotoxicity and in human induced pluripotent stem cell-derived cardiac microtissues exposed to acute doxorubicin injury. Anticancer activity was assessed across multiple cancer cell lines using 2D monolayers and 3D microtissues. ResultsIn vivo, DRP1i2 preserved left ventricular ejection fraction, reduced interstitial fibrosis and cardiomyocyte atrophy, and attenuated doxorubicin-induced myocardial proteomic remodelling. In human cardiac microtissues, DRP1i2 improved viability and restored contractile function despite persistent mitochondrial oxidative stress. DRP1i2 showed modest anticancer activity in MG63 osteosarcoma cells in both 2D and 3D systems and did not diminish doxorubicin efficacy in other cancer models (MDA-MB-231 breast, OVCAR3 ovarian, and A549 lung adenocarcinoma). Combined treatment further enhanced cytotoxicity selectively in MG63 cells. ConclusionsDRP1i2 exerts complementary cardioprotective and anticancer actions through modulation of shared mitochondrial pathways, identifying Drp1 as a druggable target in cardio-oncology. These findings support DRP1i2 as a first-in-class Drp1 inhibitor and highlight mitochondrial dynamics as a promising therapeutic axis to preserve anthracycline efficacy while reducing cardiotoxicity. Clinical PerspectivesExcessive Drp1-mediated mitochondrial fission links anthracycline cardiotoxicity with cancer cell survival. Inhibition with DRP1i2 preserved cardiac structure and function in a chronic doxorubicin cardiotoxicity model without compromising anti-cancer activity, representing mechanism-based cardioprotection, where the heart is protected by directly targeting the molecular processes driving injury. Translation will require pharmacologic profiling and testing in tumour-bearing and comorbid models, followed by early-phase trials to confirm safety and efficacy.

Crush syndrome (CS) is characterised by ischaemia/reperfusion-induced rhabdomyolysis, leading to systemic inflammation and high mortality. Building on our previous findings that intravenous nitric oxide (NO) donors improve survival in this condition, we investigated the therapeutic efficacy of inhaled NO delivered via a portable, controlled-release device in an experimental rat model of CS. Anaesthetised rats underwent bilateral hindlimb compression using rubber tourniquets for 5 h, followed by reperfusion. Among the various inhalation conditions tested, administration of NO (160 parts per million) for 2 h after reperfusion significantly increased survival rate from 20 to 90%. Improvements in haemodynamic parameters, biochemical markers, and histopathological findings correlated with enhanced survival outcomes. These results suggest that on-site NO inhalation therapy may serve as an effective first-line, emergency intervention for CS, particularly in disaster settings. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/710439v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1de2a5forg.highwire.dtl.DTLVardef@b0048eorg.highwire.dtl.DTLVardef@1fb310borg.highwire.dtl.DTLVardef@50da9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ischaemic heart disease remains the leading cause of mortality worldwide, yet no therapies prevent cardiomyocyte death during acute ischaemia-reperfusion injury (IRI). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a platform for modelling cardiac injury, but their immature phenotype limits the physiological fidelity of in vitro models. Here, we systematically evaluated how experimental variables used during preparation of hiPSC-CM endpoint assays influence cardiomyocyte maturation and susceptibility to IRI. Integrating literature mining, molecular profiling, statistical genetics, and functional assays, we examined the effects of replating conditions, backbone media, metabolic substrates, and signalling modulators. We define the relationship between culture conditions and metabolic supplements in determining contractile maturation and sensitivity to IRI. Notably, we show that metabolic composition of the backbone medium establishes the baseline maturation state and determines responsiveness to additional maturation cues. These findings identify metabolic environment as a dominant regulator of injury susceptibility and provide a framework for improving the physiological fidelity of hiPSC-CM models of cardiac ischaemia.

Liver sinusoidal endothelial cells (LSECs) play essential roles in liver regeneration after injury, but the underlying mechanisms remain incompletely defined. Here we report that leukemia inhibitory factor (LIF), which is rapidly induced after liver injury, acts as a key regulator of LSECs-driven liver regeneration through interaction with LSECs-enriched LIF receptor (LIFR). LIF directly stimulates LSECs proliferation and induces hepatocyte growth factor (HGF) release in a dose-dependent manner via LIFR signaling in LSECs, thereby indirectly promoting hepatocyte proliferation. Systemic LIF neutralization or endothelial cells (ECs)-specific Lifr loss impairs liver regeneration, whereas low-titer AAV-mediated LIF expression increases vascular density, elevates circulating HGF, and improves early liver recovery after partial hepatectomy (PHx) in mice. Together, these findings establish LIF-LIFR as a previously unrecognized endothelial axis to promote hepatocyte proliferation and suggest potential therapeutic strategies to enhance liver repair in patients. HighlightsO_LILIF is upregulated after liver injury and LIF neutralization impairs liver recovery. C_LIO_LILIFR displays the highest expression in ECs; endothelial-specific Lifr deletion delays liver regeneration after injury. C_LIO_LILIF mediates a positive feedback loop including LSECs proliferation as well as HGF release via LIFR pathway. C_LIO_LILIF overexpression increases liver-to-body weight ratio in a dose-dependent manner and accelerates liver regeneration at early stage. C_LI Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/707802v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13b42feorg.highwire.dtl.DTLVardef@1ab6390org.highwire.dtl.DTLVardef@115c157org.highwire.dtl.DTLVardef@1486993_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

BackgroundVascular mural cells (MC) are essential components of vasculature, playing critical roles in tissue regeneration and cell therapy. The use of animal derived ancillary materials, like fetal bovine serum (FBS), in the induction of MC from human pluripotent stem cells (hPSCs), represents one of the biggest limitations to guarantee preclinical safety standards required to use this products in clinical settings. This study aimed to validate human platelet lysate (hPL) as a serum-free alternative for MC differentiation from hPSCs. MethodsComparison of MC differentiation efficiency from hiPSC using FBS vs hPL supplemented cultures was performed, along with functionality and gene expression assessment through bulk RNA sequencing. ResultsOptimization of hPL concentration identified hPL1% as the most effective condition, yielding PDGFR-{beta}+/CNN1+ MC, with a comparable efficiency to FBS10% and similar interaction with endothelial cells in vascular formation assays. However, distinct transcriptional profiles revealed that FBS10% and hPL1% drive differentiation toward different MC subphenotypes; hPL1% promoted contractile gene expression, while FBS10% enriched extracellular matrix pathways. Higher hPL concentrations further shifted differentiation toward cardiomyocytes. ConclusionIn monolayer in vitro differentiation of MC from hiPSC, the differentiation efficiency using hPL 1% supplementation is equivalent to FBS 10%, while supporting a more contractile phenotype. These findings establish hPL as a xeno-minimized, clinically compliant substitute for FBS for hPSC-derived MC differentiation, an important breakthrough for regenerative medicine.

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.

Cardiac fibrosis is a pathological process in which the myocardium stiffens due to the overproduction of extracellular matrix (ECM) proteins. Cardiac fibroblasts activate to myofibroblasts in response to the inflammatory cytokine transforming growth factor beta1 (TGF-{beta}1) to promote fibrotic scarring. Biological sex also influences cardiac fibrosis progression and patient outcomes, where males exhibit increased fibrotic scarring after acute inflammation relative to females. At the cellular level, sex differences in TGF-{beta}1-mediated cardiac myofibroblast activation processes have not been clearly defined. We hypothesized that TGF-{beta}1 would cause sex-specific cardiac myofibroblast activation levels and alter the secretion of bioactive molecules to modulate sex differences in cardiac fibrosis. Primary left ventricle cardiac fibroblasts were isolated from male and female C57BL/6J mice and cultured on hydrogel biomaterials mimicking native myocardial ECM stiffness and treated with TGF-{beta}1 and/or the TGF-{beta}1 receptor inhibitor SD208. Male myofibroblasts exhibited increased -SMA stress fiber formation, increased SMAD2/3 localization, and greater resistance to SD208 inhibition compared to female myofibroblasts on hydrogels at various time points tested. Sex differences in relative secreted cytokine abundance were also determined, with male CFs secreting increased vascular endothelial growth factor (VEGF) and female CFs producing increased periostin and fibroblast growth factor 21 in response to TGF-{beta}1. Our findings establish that TGF-{beta}1 mediates sex differences in cardiac myofibroblast activation on hydrogels and secreted factors that may modulate the myocardial microenvironment. Our work underscores the importance of using hydrogels as cell culture platforms to recapitulate sex-specific cardiac fibrosis phenotypes as a steppingstone towards identifying sex-dependent therapeutic interventions for cardiac fibrosis.

Mitochondrial dysfunction is a pervasive hallmark of diverse diseases. In endothelial cells (ECs), oxidative stress, bioenergetic failure, and dysregulated mitochondrial dynamics (fusion-fission, mitophagy) damage the endothelium and promote vascular pathologies such as diabetes, atherosclerosis, and aging. Mitochondrial augmentation, via direct transplantation of isolated mitochondria or cell-to-cell transfer of the organelle, has emerged as a strategy to restore mitochondrial function in metabolically compromised cells. We recently established that overexpressing nuclear respiratory factor 1 (NRF1), a driver of mitochondrial biogenesis, in mesenchymal stem cells (MSCs) increases mitochondrial content and preserves mitochondrial function under senescence-inducing stress. Here, we advance NRF1-primed MSCs as enhanced mitochondrial hubs for intercellular mitochondrial delivery to cells undergoing mitochondrial dysfunction. We hypothesized that NRF1 overexpression engages mitochondrial transfer machinery, thereby enhancing both tunneling nanotube (TNT)- and extracellular vesicle (EV)-mediated mitochondrial transfer to stressed ECs, improving EC mitochondrial fitness and health. mRNA-mediated NRF1 priming of MSCs increased expression of proteins involved in mitochondrial motility and transfer, enhanced TNT formation, and increased production of mitochondria-containing EVs. Single-cell RNA sequencing (scRNA-seq) results show that NRF1 priming shifted MSCs into distinct transcriptional states, with NRF1-enriched clusters exhibiting coordinated upregulation of cell-adhesion/cytoskeletal connectivity programs and vesicle-fusion/trafficking pathways, features consistent with enhanced structural coupling and secretory transfer capacity. NRF1 priming increased TNT-like F-actin intercellular bridges in direct co-culture and elevated mitochondria-containing EV transfer in transwell assays, demonstrating augmented mitochondrial delivery through both contact-dependent and contact-independent routes. Consequently, recipient ECs displayed reduced mitochondrial ROS, preserved membrane potential, improved oxidative phosphorylation and ATP production, rebalanced mitochondrial dynamics of fusion-fission and mitophagy. NRF1-primed MSCs further attenuated oxidative stress-induced EC senescence and apoptosis. Together, these findings identify NRF1 activation as a mechanism to reprogram MSCs into high-capacity mitochondrial donors and support NRF1-driven mitochondrial hub engineering as a strategy to strengthen mitochondrial transfer-based therapies for diseases characterized by mitochondrial dysfunction.

BackgroundEndoplasmic reticulum (ER) stress and ER-mitochondria Ca2+ dysregulation contribute to cardiomyocyte injury, yet endogenous regulators at ER-mitochondria interfaces that restrain this cascade remain poorly defined. Pannexin 2 (Panx2), the most structurally divergent pannexin isoform, has been implicated in stress response, but its cardiac localization and function are unclear. MethodsPanx2 localization and function were assessed in human AC16 cardiomyocytes using high-resolution confocal imaging and complementary loss- and gain-of-function approaches during thapsigargin-induced ER stress, with validation in adult mouse ventricular cardiomyocytes. ResultsPanx2 localizes predominantly to the ER and mitochondria-associated membranes, rather than the plasma membrane. Panx2 knockdown reduced ER Ca2+ stores and increased basal cytosolic and mitochondrial Ca2+. During ER stress, Panx2 deficiency markedly amplified Ca2+ dysregulation, mitochondrial dysfunction, unfolded protein response activation, and cytotoxicity, with PERK-dominant signaling and increased IRE1a activation. Notably, PERK inhibition preferentially rescued the Panx2-deficient phenotype, providing the greatest improvement in viability and reduction in cytotoxicity. Panx2 deficiency also enhanced inflammasome/ pyroptotic signaling via the NLRP3-caspase-1-gasdermin D axis. Conversely, Panx2 overexpression suppressed PERK activation and attenuated ER stress-induced injury. Panx2 ablation similarly sensitizes adult ventricular cardiomyocytes to ER stress. ConclusionsPanx2 functions as an organelle-associated checkpoint at ER-mitochondria interfaces that stabilizes Ca2+ homeostasis and limits PERK-dominant ER stress signaling and inflammatory cell death programs in cardiomyocytes, providing a mechanistic framework for cardiomyocyte loss in cardiac disease. Research PerspectiveO_ST_ABS1. What New Question Does This Study Raise?C_ST_ABSDoes Panx2 serve as an endogenous "stress threshold" determinant in cardiomyocytes in vivo, governing when ER stress transitions from adaptive signaling to PERK-driven mitochondrial failure and inflammasome-associated inflammatory cell death during cardiac injury (e.g., ischemia-reperfusion, pressure overload, or cardiometabolic stress)? 2. What Question Should Be Addressed Next?In clinically relevant models of heart disease (ischemia-reperfusion), test whether cardiomyocyte-specific Panx2 loss or augmentation alters infarct size, arrhythmia burden, ventricular remodeling, and functional recovery, and determine whether targeting the Panx2-PERK axis (e.g., selective PERK modulation in the acute reperfusion window or Panx2-directed strategies) reduces cardiomyocyte loss without impairing adaptive stress signaling needed for repair.

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.