npj Regenerative Medicine

Large full-thickness (LFT) skin wounds remain a major clinical challenge, and progress in regenerative medicine has been limited by poor translation from animal models to humans. A key limitation is that commonly used species such as mice, rats, and rabbits are loose-skinned, whereas humans are tight-skinned with distinct skin architecture. Although pigs more closely resemble human skin, widely used breeds have lost secondary (vellus-like) hair follicles through artificial selection, restricting their utility for studying ectodermal organ regeneration. Here, we characterize the development, patterning, and molecular features of secondary hair follicles in the Lanyu pig (Sus scrofa taivanus), an indigenous breed that retains these structures. Whole-mount and histological analyses revealed two distinct follicle populations: primary follicles arranged in stable triplet clusters and smaller secondary follicles distributed interstitially. A developmental time course using alkaline phosphatase (ALP) staining identified sequential stages of secondary follicle morphogenesis--placode, hair germ, hair peg, and mature follicle--occurring after primary follicle establishment. Immunohistochemical analysis demonstrated conserved epithelial- mesenchymal interactions, progressive epithelial stratification, and dynamic {beta}-catenin signaling during secondary follicle development. Keratin expression patterns and follicular architecture closely resembled those of human vellus hair follicles, supporting the translational relevance of this model. Notably, secondary follicles were retained into adulthood, and genetic analyses of outcrossed animals suggest that this trait follows an autosomal dominant inheritance pattern. Together, these findings establish the Lanyu pig as a tight-skinned mammalian model that preserves vellus-like hair follicles, providing a platform for investigating hair follicle-mediated skin regeneration and improving translational relevance for human wound healing.

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

Objective: To evaluate the effect of residual hair (RH) biomaterial particulates, biphasic electrical stimulation (ES), and their combination (RHES) on the kinetics and quality of skin wound healing. Method: Eighteen adult albino mice received bilateral, splinted 10-mm full-thickness dorsal excisional wounds and were randomly assigned to one of three animal groups producing four wound-level treatment conditions: untreated control (-) (n = 12), RH (n = 12), ES (n = 6), and combined RHES (n = 6 wounds). Daily wound images were segmented using an AI-assisted workflow: a U-Net (ResNet34 encoder, ImageNet-pretrained, trained on a parallel single-expert tracing study with held-out validation Dice = 0.906) generated initial boundary predictions, each reviewed and corrected as needed. Wound size measures (perimeter, area, equivalent diameter [D_eq], circularity, aspect ratio) were normalized to the day-0 value of each wound and analyzed by linear mixed-effects regression with mouse identity as a random intercept and mouse body weight as a covariate. On day 7, wounds were excised, fixed, processed for histology, and analyzed by Masson's trichrome (collagen content in granulation tissue) and GAP-43 immunohistochemistry (a marker of regenerative cellular activity). Results: All three treatments significantly accelerated wound closure compared to (-) (Day x Treatment interaction {chi}2(3) = 36.4, ***p < 0.0001). The closure-rate advantages on the log-D_eq scale were ES -0.047/day (***p < 0.0001), RHES -0.029/day (***p = 0.0005), and RH -0.022/day (**p = 0.0015). By day 7, mean D_eq had decreased to 0.58 of the day-0 value in ES, 0.69 in RHES, 0.73 in RH, and 0.79 in (-). Tissue analyses revealed treatment-specific differences in healing quality: RH and RHES wounds contained 6.1x and 8.5x more collagen in granulation tissue than (-) (both **p = 0.002 vs (-); both **p = 0.009 vs ES), and showed approximately 16x and 27x greater mean GAP-43 expression than (-), respectively; the RHES increase remained significant after Bonferroni correction (adjusted *p = 0.042), whereas the RH increase did not (adjusted p = 0.058). ES alone did not significantly increase either collagen content or GAP-43 expression. Wound shape was more circular and more stable across days in RH-containing groups. Mouse body weight did not predict closure, whereas image-derived dryness, eschar coverage, and wound contraction were significant negative predictors of measured wound size. Conclusion: ES, RH, and RHES each significantly improve wound closure kinetics. The improvement appears mechanistically distinct: ES principally accelerates closure rate, while RH principally enhances tissue-level regenerative markers (collagen deposition and GAP-43 expression). RHES combines both advantages.

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

Fibro/adipogenic progenitors (FAPs) are multipotent stromal cells that support myofiber regeneration, but can also give rise to intramuscular adipose tissue (IMAT) and fibrotic scar tissue. While the Hedgehog pathway suppresses FAP adipogenesis and promotes myofiber repair through ligand Desert Hedgehog, the key cell type that senses this signal has remained unclear. Here, we demonstrate through FAP-specific deletion of the Hedgehog signal transducer Smoothened that FAPs are the primary Hedgehog-responding cells during muscle regeneration. Loss of Smoothened in FAPs increases IMAT, causes persistent fibrosis, reduces the Hedgehog-dependent effectors TIMP3 and GDF10, and impairs myofiber regeneration. FAPs lacking Smoothened also fail to support in vitro myoblast differentiation and fusion as efficiently as control FAPs, showing that Hedgehog signaling helps establish a pro-myogenic FAP state early after injury. Pharmacological Hedgehog activation via the Smoothened agonist SAG fails to rescue adipocyte accumulation or myofiber regeneration when FAPs lack Smoothened. Together, these findings provide direct genetic evidence that FAPs are the primary cellular mediators of Hedgehog signaling in muscle and establish FAP Hedgehog signaling competence as a key determinant of regenerative outcome and a target for restoring muscle repair in disease.

Adult skin normally resolves injury through rapid closure and fibrotic matrix deposition, often at the cost of permanent appendage loss. We tested whether spatially controlled microtrauma could instead serve as a regenerative entry point when paired with temporally coordinated molecular cues. We engineered a hierarchical extracellular-matrix-based microneedle patch that combines rapid local availability of verteporfin, an inhibitor of YAP-associated mechanotransduction, with sustained retinoic-acid delivery to support follicle-regenerative signalling. The microneedle interface was evaluated in full-thickness rabbit ear wounds, which are prone to hypertrophic scarring, and in Bama miniature-pig wounds, whose skin architecture more closely resembles human skin. Across both models, staged dual-cue treatment accelerated wound closure, reduced collagen-dense scar formation and promoted the appearance of hair-bearing tissue and histologically identifiable follicular structures. These findings support a trauma-guided regeneration framework in which controlled microinjury is used not only for delivery but also to open a transient repair niche that can be molecularly redirected toward appendage-bearing skin restoration. ImportanceMicroneedles are generally treated as minimally invasive delivery devices. Here, the microinjury itself is incorporated into the therapeutic design. The study provides cross-species proof of concept that a patterned injury interface, combined with staged anti-fibrotic and pro-regenerative signalling, can shift wound repair away from fibrotic closure and toward hair-follicle-containing skin. This concise preprint reports the central concept and the rabbit and porcine evidence supporting it; expanded mechanistic and source datasets will be reported separately.

BackgroundPhysical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. MethodsPublicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88fl/fl) and wild-type controls (Ift88fl/fl) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro-computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Students t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. ResultsHere, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. ConclusionsOur results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.

BackgroundTissue vascularization relies on the organotypic specification of endothelial tip cells to interpret local cues and guide angiogenic sprouts into specific tissue compartments. However, the molecular regulators controlling brain and retinal endothelial tip cell identity remain poorly understood. MethodsEndothelial-specific Sox9 loss-of-function mouse was combined with single-cell, spatial, and bulk RNA sequencing analyses of developing mouse brain and retinal vasculature, as well as experimental ischemic stroke. Transcriptomic findings were validated using in situ hybridization, immunofluorescence, and functional angiogenesis assays. ResultsTranscription factor activity analysis of single-cell RNA-sequencing datasets identified SOX9 as a candidate regulator selectively enriched in developing brain and retinal endothelial tip cells. Endothelial-specific deletion of Sox9 impaired brain and neuroretina vascularization and disrupted the tip cell transcriptomic program, resulting in reduced sprouting angiogenesis and matrix-remodeling pathways. Conversely, SOX9 overexpression in HUVECs promoted neuro-tip-like signatures and enhanced endothelial invasion and sprouting. Following ischemic stroke, single-cell and spatial transcriptomic analyses identified a transient angiogenic endothelial population within the ischemic area. However, these cells failed to express Sox9 and lacked key developmental brain tip cell features. ConclusionsSOX9 is a key regulator of endothelial tip cell identity and neuronal angiogenesis. These findings reveal fundamental differences between developmental and injury-induced vascular responses and identify SOX9 as a potential therapeutic target to promote functional vascular regeneration.

Peripheral nerve injuries often result in prolonged skeletal muscle denervation, leading to progressive atrophy, fibrosis, neuromuscular instability, and loss of regenerative capacity before axons can reinnervate distal targets. Here, we developed a non-viral strategy using tissue nanotransfection (TNT) to deliver the neurogenic transcription factor cocktail Ascl1, Brn2, and Myt1l (ABM) directly to denervated skeletal muscle. In vitro, ABM-transfected myoblasts sustained expression of the reprogramming factors, acquired neuron-like morphologies, upregulated neuronal markers including Tuj1, Map2, and Syp, and exhibited electrophysiological properties consistent with membrane excitability. RNA sequencing confirmed broad activation of neurogenic transcriptional programs, with enrichment of pathways associated with neuronal fate commitment, neuron differentiation, axon guidance, synaptogenesis, and developmental signaling. In a mouse model of sciatic nerve transection, TNT enabled localized ABM expression in denervated gastrocnemius muscle. ABM-TNT treatment accelerated resolution of denervation-associated fibrillation potentials and showed trends toward improved twitch and tetanic torque, compound muscle action potential amplitudes, and muscle mass preservation. Transcriptomic profiling of treated muscles 5 weeks after injury revealed distinct gene expression programs enriched for muscle regeneration, neuromuscular organization, trophic support, extracellular matrix remodeling, angiogenesis, myogenesis, and metabolic adaptation. Network analyses further identified activation of neurogenic regulators, neurotrophic signaling, and vascular-support pathways. These findings establish TNT-mediated ABM delivery as a non-viral platform for inducing neurogenic and myoprotective programs in denervated muscle, suggesting a potential strategy to preserve muscle viability during the prolonged interval required for peripheral nerve regeneration.

Human cardiac injury responses are governed by dynamic interacting processes that are difficult to resolve. Unlike adults, fetal mammalian hearts regenerate through coordinated remodeling and proliferation supported by a pro-regenerative immune environment, extracellular matrix (ECM), and immature cardiomyocytes, including trabecular subtypes. Here, we establish a modular human cardioid injury platform to dissect these interactions. We show that anti-inflammatory macrophages selectively migrate to the injury, clear debris, and promote ECM remodeling, whereas inflammatory macrophages suppress cardiomyocyte proliferation. Synergistic FGF2-NRG1 signaling induces trabecular identity and morphology in a hyaluronan-dependent manner, conferring enhanced injury repair, characterized by cytoskeletal remodeling and cardiomyocyte proliferation mediated by YAP and WNT signaling. Exogenous YAP, but not WNT, is sufficient to promote repair in non-trabecular cardioids. These findings uncover coordinated immune-ECM-cardiomyocyte interactions governing human fetal regenerative competence and mechanistically resolve remodeling and proliferative components of cardiac repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/735236v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7ff079org.highwire.dtl.DTLVardef@184d5bdorg.highwire.dtl.DTLVardef@1ec775borg.highwire.dtl.DTLVardef@190008e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ability to induce tissue regeneration on demand using biomaterials remains a major goal in biomedical research, yet significant challenges persist. Among the most advanced biomaterial models, the nanofiber-hydrogel composite has demonstrated a striking ability to induce soft adipose tissue remodeling at the injection site without incorporating exogenous biological cues.1,2 However, the underlying mechanisms that drive such a tissue response remain unclear. Here, we show that biomaterial-induced tissue remodeling is driven by sustained and controlled inflammation mediated by macrophages in strong communication with fibroblasts. Notably, both pro-inflammatory and anti-inflammatory signals remained elevated during this process in the long-term, challenging the prevailing notion that inflammation opposes remodeling. Using macrophage depletion in mice, we demonstrate that macrophages are essential for this process. Single-cell RNA sequencing further revealed robust fibroblast-to-macrophage signaling, contrasting with the conventional macrophage-to-fibroblast paradigm, and identified unique Spp1 macrophages and Ctla2a fibroblasts within the remodeling niche. These findings provide a comprehensive view of the immune landscape in biomaterial-induced tissue remodeling, highlighting key cellular interactions, prolonged kinetics, and unexpected signaling pathways. By defining key targets and fundamental principles, this work has broad implications for advancing biomaterial-induced tissue regeneration.

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Long-term ex situ machine perfusion of donor organs is an emerging clinical strategy that creates a window for advanced therapeutic interventions. Replacing donor endothelial cells during machine perfusion with recipient endothelium could conceal the allogeneic epithelium from the recipients (humoral) immune response. We postulated that brief exposure to low concentrations of decellularizing agents could selectively remove vascular endothelium. Porcine kidneys were partially decellularized with five 2-minute infusions of either 0.01%, 0.1% or 1.0% SDS during acellular perfusion. As proof-of-concept, fluorescently-labelled porcine endothelial colony forming cells (ECFCs) were infused into the renal vein and artery of a partially decellularized kidney. Tissue analysis identified 0.1% SDS effectively removed endothelial cells from glomerular and peritubular capillaries. Infused ECFCs could be found back within the glomeruli. However, partial decellularization significantly impaired renal flow and increased vascular resistance. While partial decellularization successfully removed donor endothelium, the loss of vascular patency limits its clinical potential. Future research should prioritize modifying rather than removing donor endothelial cells.

Myocardial development requires precise modulation of its growth and maturation by the mechanical loads within cardiac cycle, including preload and afterload. However, the mechanisms by which these natural cardiac loads interact to simultaneously govern growth and maturation of the developing myocardium in both cellular and mesoscale levels remain poorly understood. Here, we developed a naturally engineered fetal ventricular tissue (NFVT) platform that enables the application of afterload under dynamic preload using cyclic stretching with an asymmetrical duty cycle (asymmetrically cycled preload) to better replicate the natural cardiac loading in chick NFVT. Our results showed that low afterload (LA) enhanced NFVT contractile function with sustained tissue growth and improved cardiomyocyte maturation while suppressing fibrosis phenotypes. These effects were associated with reduced YAP1 and NOTCH activation in cardiomyocytes and enhanced tissue architecture. In contrast, high afterload (HA) induced contractile impairment with fibrotic remodeling through activation of cardiomyocyte PIEZO1/YAP1 signaling and sustained fibroblast entanglement. TeaserLow afterload under asymmetrically cycled preload promotes NFVTs contractile function with sustained tissue growth and cardiomyocyte maturation while suppressing fibrosis phenotypes through regulation of cellular mechanotransduction, including minimal PIEZO1 expression and inhibited YAP1 and NOTCH activation in cardiomyocytes, and improvement of collective cellular organization.

Islet transplantation can restore glycemic control in type 1 diabetes, yet the heterogeneity of patient immune responses and transplant outcomes motivates the need for technologies to monitor the graft. Since transplanted islets are not readily accessible for biopsy due to their diffuse engraftment within the liver, clinical monitoring relies on measurements such as islet mass, blood glucose, and C-peptide levels, which are lagging indicators that change only after substantial graft injury. Here, we developed a minimally invasive synthetic immunological niche (IN) that captures graft-associated immune responses through serial subcutaneous biopsy. We evaluated the IN across murine syngeneic, allogeneic, and autoimmune islet transplant models, including CD40/CD154 costimulatory blockade with anti-CD40L. In syngeneic versus allogeneic recipients, IN identified immune populations and transcriptomic signatures that mirrored the graft and distinguished healthy from rejecting grafts. In anti-CD40L treated allografts, IN revealed innate macrophage- and dendritic cell-associated programs linked to graft acceptance versus rejection, whereas IN from untreated allografts showed stronger adaptive immune signatures. Longitudinal IN profiling further detected progressive inflammatory activation in accepted allografts, indicating persistent subclinical risk. Finally, in an autoimmune allograft model treated with anti-CD40L plus rapamycin, IN identified a 13-gene signature that separated early from late rejection trajectories and distinguished autoimmune-from alloimmune-associated rejection programs. Overall, these findings establish IN as a surrogate tissue for minimally invasive monitoring of islet graft and early detection of rejection-associated immune dysregulation. One Sentence SummaryAn engineered immunological niche captures distinct immune signatures of allo- and auto-mediated islet transplant rejection

Artery endothelial cells (ECs) arise through different pathways, including differentiation from mesodermal cells (vasculogenesis) or from already established vein or capillary plexus ECs (angiogenesis), the latter being most common during embryonic development and regeneration. Understanding the vein-to-artery (v2a) transition could improve revascularization therapies, but progress is limited by a lack of human models. Here, we develop a human pluripotent stem cell (hPSC) differentiation protocol that models the v2a EC conversion. Comparing v2a and mesoderm-to-artery (m2a) transcriptomes with publicly available single cell RNA sequencing (scRNA-seq) data from human embryos showed they reflected angiogenesis- and vasculogenesis-derived artery ECs, respectively. This reductionist system revealed that VEGF activation alongside PI3K inhibition was sufficient for vein ECs to acquire arterial identity within 48 hours. We model a critical step in vascular development and define the minimal signals required for artery differentiation from veins, providing a framework to promote this conversion in revascularization or therapeutic contexts.

Bioengineered, transplantable kidney tissue using decellularized scaffolds offers a promising strategy to overcome the shortage of donor kidneys that limits organ transplantation for patients with end stage renal disease. These kidney scaffolds retain essential extracellular matrix architecture, providing a biologically active niche for recellularization. Successful generation of bioengineered kidney tissues includes enhanced patent vasculature and mature, functional nephrons with collecting ducts. Here, we report the development of engineered kidney tissue consisting of reconstituted kidney scaffolds and human pluripotent stem cell-derived nephron and ureteric bud progenitors. Structural analysis of recellularized kidney scaffolds showed advanced nephron structures that became more mature and exhibited interconnected nephron and collecting ducts. In vivo engraftment of reconstituted kidney scaffolds in mice led to vascularization, maturation, and secretory function. Notably, mouse-graft vascular anastomosis was evident with erythrocytes present in vasculature and nephron-secreted proteins detected in mouse urine, indicating functional integration. This approach demonstrates the feasibility to generate advanced bioengineered kidney tissues that offer a versatile platform for disease modeling, drug screening, and regenerative medicine.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have become defining therapies in the management of type 2 diabetes and obesity. Despite recent interest in the effects of GLP-1RA therapy on skeletal muscle, their influence on muscle repair after injury remains largely untested. Because GLP-1RA use is common in populations at heightened risk for diminished regenerative capacity, a critical unanswered question is whether GLP-1R agonism supports muscle regeneration or alters the normal course of recovery after injury. Using intramuscular glycerol injection as an adipogenic injury model, we assessed whether semaglutide, a widely prescribed GLP-1RA, alters the balance between myogenesis and adipogenesis during regeneration. Surprisingly, semaglutide treatment markedly increased the formation of intramuscular adipose tissue (IMAT) and inhibited the growth of regenerated fibers. These effects were injury-dependent, as uninjured muscle showed no detectable differences in IMAT or myofiber size. Together, these findings identify a previously underappreciated context in which GLP-1RA therapy may adversely affect muscle quality.

Heart transplantation (HT) is the durable therapy for end-stage heart failure (HF). Despite advances in immunosuppression, cardiac allograft vasculopathy (CAV) remains a leading cause of late graft failure and mortality in the modern era. Prior studies have established donor age and immunological phenomena, such as acute cellular rejection (ACR), antibody-mediated rejection (AMR), and development of donor-specific antibodies (DSAs) as risk factors for CAV. However, it remains unclear whether acute rejection (AR) that occurs early post-HT, when individuals experience the highest degree of immunosuppression, reflects higher baseline immune activity and confers a higher risk of future CAV compared to later AR, when immunosuppression is minimized. We therefore examined whether AR occurring during pre-specified early and intermediate intervals compared to those who did not experience AR in the first post-HT year was associated with future CAV among recipients without CAV at 1 year.

Eya3 and Eya4 are two Eya genes expressed in adult myogenic stem cells, where they may act as SIX cofactors. We analyzed muscle regeneration in single and compound Eya3 and satellite cell-specific Eya4 mutant mice. A kinetic analysis of muscle regeneration after Notexin injury of the Tibialis Anterior revealed no major phenotype at 4, 14, and 30 days after injury in terms of PAX7+ cell number and myofiber cross-sectional area in Eya3 mutants, while all parameters were decreased in Eya4 mutants and further worsened in Eya3/Eya4 double mutants, in which we also observed a modification of the myofiber phenotype at 30 days after injury. Satellite cells were cultured ex vivo and Eya4 deletion was induced by Ad-Cre-mediated recombination. While single Eya3 mutant cells showed normal proliferation and differentiation, double mutant cells exhibited normal proliferation but failed to fuse. Analysis of their transcriptome revealed that the expression of Myomixer, Follistatin, and Noggin was severely downregulated specifically in double mutant cells, explaining their fusion deficiency. To gain a better understanding of the involvement of Eya genes during embryonic development and the genesis of PAX7+ myogenic stem cells, we analyzed Eya1 / ;Eya2 / , Eya3 / , Eya4 / , and Eya3 / ;Eya4 / E18.5 mutant fetuses at the limb and craniofacial levels. In Eya1 / ;Eya2 / fetuses, we confirmed the absence of distal limb muscles and observed reduced craniofacial muscles. In Eya3 / ;Eya4 / fetuses, craniofacial myogenesis appeared preserved and PAX7+ myogenic stem cells were present. BackgroundThe Eyes absent (Eya) genes encode transcriptional co-activators and phosphatases that function within the PAX-SIX-EYA-DACH (PSED) regulatory network. In skeletal muscle, EYA proteins cooperate with SIX homeoproteins to control myogenic gene expression during both embryonic development and adult regeneration. While Eya1 and Eya2 are predominantly expressed in embryonic myogenic progenitors and Eya3 and Eya4 are the dominant paralogs in adult satellite cells (SC), the specific and redundant contributions of individual family members to myogenesis remain poorly characterized. MethodsWe analyzed compound Eya mutant mice during adult Tibialis anterior muscle regeneration and during embryogenesis. We complemented this analysis by performing ex vivo myogenic stem cell cultures from compound Eya mutants and examining their fusion capacity. ResultsAnalysis of muscle regeneration following Notexin injury revealed that Eya2 and Eya3 single mutants display no major regenerative deficit. In contrast, satellite cell-specific deletion of Eya4 (Eya4sc/sc) caused a transient impairment of early regeneration, with reduced numbers of smaller regenerating MYH3+ (embryonic myosin heavy chain) myofibers and a transient decrease in SC number at 4 days post-injury (dpi). Compound Eya3-/-;Eya4sc/scdouble mutants showed a more severe and persistent phenotype, with decreased myofiber cross-sectional area, reduced myonuclear accretion, accumulation of PAX7+ cells associated with regenerated myofibers, and altered fiber-type composition at 14 and 30 dpi. Ex vivo analysis of double mutant SCs revealed a specific and complete blockade of myogenic fusion without defects in proliferation or MYOD expression. Transcriptomic analysis identified severe downregulation of Myomixer, Noggin, and Follistatin in differentiating Eya3-/-;Eya4-/- SCs. Open-access SIX1 and SIX4 ChIP-seq publicly available data confirmed direct binding at the Myomixer, Noggin, and Follistatin loci, supporting a direct SIX-EYA transcriptional mechanism. In parallel, embryonic analysis demonstrated that Eya1-/-;Eya2-/-E18.5 fetuses lack distal limb musculature and display severe craniofacial muscle hypoplasia, while in Eya3-/-;Eya4-/-fetuses limb and craniofacial musculature developed with no detectable defects. ConclusionsThese results reveal distinct temporal requirements for EYA proteins in skeletal muscle: EYA1 and EYA2 are essential SIX cofactors for embryonic myogenic fate acquisition in hypaxial and craniofacial progenitors, while EYA3 and EYA4 act redundantly in adult satellite cells to enable myogenic fusion by maintaining BMP antagonist expression and Myomixer activation downstream of the SIX-EYA transcriptional complex.