npj Regenerative Medicine

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.

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.

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

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.

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.

The African spiny mouse (Acomys cahirinus) exhibits a unique capacity among mammals for scarless tissue regeneration, making it a compelling model for investigating the cellular mechanisms underlying regenerative healing. To determine how cellular heterogeneity and specific phenotypes influence fibroblast behavior, we established an immortalized Acomys fibroblast line along with a CRISPR/Cas9-mediated Col3A1 knockout variant and a DNA damage-induced senescent population. Compared with Mus musculus, NIH 3T3 fibroblasts, Acomys cells displayed distinct morphology, similar migration speeds, reduced directional persistence, and greater biophysical heterogeneity. While previous studies have linked regenerative wound healing to the elevated expression of collagen type III (Col3A1), CRISPR-mediated knockout of Col3A1 in Acomys fibroblasts yielded comparable biophysical profiles to wild-type cells in 2D culture. To examine additional contributors to the enhanced wound-like matrix environment, we established a senescence model in which Acomys fibroblasts exhibited elevated resistance to DNA-damaging agents, complete loss of proliferation, and altered single-cell morphology. In 3D collagen gel contraction assays, Col3A1 knockout attenuated matrix remodeling capacity, whereas the introduction of a small fraction of senescent cells enhanced gel contraction and remodeling dynamics, suggesting that senescent fibroblasts can modulate collective matrix behaviors. Together, these findings demonstrate that both Col3A1 expression and senescence-associated cell states contribute to fibroblast-driven matrix remodeling, highlighting Acomys fibroblasts as a valuable model for investigating how cellular heterogeneity and senescence-associated cell phenotypes could influence regenerative wound healing.

Liver regeneration is initiated by rapid vascular changes, yet how blood flow-derived mechanical cues are decoded by liver sinusoidal endothelial cells (LSECs) remains unclear. Here, we found that partial hepatectomy generates temporally distinct mechanical cues in vivo, with a transient rise in shear stress followed by progressive sinusoidal dilation and endothelial stretch. To dissect these forces, we developed a liver regeneration chip that reconstructs sinusoidal architecture and enables independent or coupled manipulation of shear stress and mechanical stretch. Shear-dominant, stretch-dominant, and coupled mechanical modalities induce divergent LSEC regenerative programs involving extracellular matrix remodeling, cell-cycle regulation, cytoskeletal organization, and angiocrine signaling. Mechanistically, force-specific pathways, including Wnt, HIF-1, NF-{kappa}B, and Piezo1-associated signaling, mediate these outputs. Inhibition of these pathways after partial hepatectomy impairs hepatocyte proliferation and survival. These findings reveal that LSECs temporally decode blood flow-derived mechanical forces into distinct regenerative outputs, establishing endothelial mechanotransduction as an upstream regulator of liver regeneration. HIGHLIGHTS{blacksquare} Partial hepatectomy decouples transient shear from progressive stretch in vivo. {blacksquare}A liver regeneration chip recreates structure and distinct mechanical modalities in sinusoids. {blacksquare}Distinct mechanical modalities encode divergent LSEC regenerative programs. {blacksquare}Force-specific LSEC mechanotransduction supports hepatocyte proliferation and survival.

An autocrine pro-adhesive/pro-contractile signaling loop, through the mechanosensitive transcriptional cofactor YAP, promotes fibrosis. The CCN family of matricellular proteins modify adhesive signaling. Of these, CCN3 is antifibrotic. We show that BLR-200, a CCN3-derived peptide, has anti-fibrotic properties in the bleomycin-induced model of scleroderma skin fibrosis. In vitro, BLR-200 delayed, but did not abolish, fibroblast adhesion to collagen and nuclear YAP localization. In vivo, BLR-200 prevented/treated bleomycin-induced skin fibrosis, and reduced bleomycin-induced expression of profibrotic genes including alpha-smooth muscle actin, CCN1 and CCN2. Lineage tracing and scRNA-seq analyses revealed that the myofibroblasts in this model were quantitatively derived from collagen-lineage Pi16+/Col15+ve fibroblasts. BLR-200 prevented myofibroblast differentiation in this model and trajectory of fibroblasts toward a Sfrp2-positive subset, a cell type associated with poor clinical outcome. BLR-200 impairs YAP activation in vitro and appearance of translationally-relevant fibroblast subtypes in vivo and is a novel anti-fibrotic agent for SSc skin fibrosis.

Background & AimsThe human esophagus undergoes a tightly regulated developmental program, transitioning from a simple columnar epithelium in early development to a mature stratified squamous tissue essential for adult barrier function. Here, we constructed a developmental cell atlas spanning early development to adulthood and leveraged it to generate physiologically relevant in vitro models. MethodsWe utilized single-cell RNA sequencing and spatial multiplex proteomics of human esophageal tissue from early development through adulthood. We established a feeder-supported 2D culture system and a Matrigel-free, suspension-based 3D esophagoid model in a 96-well format. To interrogate WNT2B function, we analyzed patient tissue harboring WNT2B loss-of-function mutations and performed WNT inhibition in esophagoids. ResultsSequencing profiling identified stage-specific epithelial populations: multiciliated and GPC3 basal cells were unique to early development; KRT14 basal and CRNN luminal cells were adult-specific; and COL17A1, LY6D, and KRT4 populations were shared across stages. Spatially organized WNT2B, KIT, and VWC2 mesenchymal subtypes were identified. The 2D system preserved both epithelial and mesenchymal compartments with transcriptional fidelity. Esophagoids exhibited basal-to-luminal stratification, mesenchymal compartmentalization, and required stromal interactions for formation. WNT2B repressed self-renewal of TP63 basal progenitors and inhibited proliferation, confirmed by pharmacologic inhibition of WNT in the in vitro esophagoids. ConclusionsWe present a stage-resolved atlas of human esophageal development and a scalable esophagoid platform recapitulating esophageal architecture. WNT2B regulates progenitor dynamics by restraining basal cell self-renewal. Esophagoids provide a physiologically relevant system for modeling esophageal development and disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733451v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1af5743org.highwire.dtl.DTLVardef@8a061dorg.highwire.dtl.DTLVardef@1975c3forg.highwire.dtl.DTLVardef@292ce9_HPS_FORMAT_FIGEXP M_FIG C_FIG Key Findings and ImplicationsO_LIDevelopmental Atlas: The study presents a comprehensive transcriptional and structural atlas of the human esophageal epithelium, identifying conserved and stage-specific epithelial populations from early development to adulthood. Notably, stage-specific gene expression of multiciliated and GPC3 basal cells were unique to early development, while KRT14 basal and CRNN luminal cells were adult specific, with COL17A1+ (basal), LY6D+ (epibasal), and KRT4+ (middle), shared at all stages. C_LIO_LIMesenchymal Diversity: Spatial and transcriptional profiling revealed distinct mesenchymal subtypes, including WNT2B, KIT, and VWC2 populations, which are spatially organized and contribute to epithelial-mesenchymal signaling. These findings reinforce the role of stromal-epithelial interactions in esophageal development. C_LIO_LI2D Esophagus Cell Culture System: A feeder-supported 2D cell culture system was developed that retains both epithelial and mesenchymal populations, preserving transcriptional fidelity and enabling long-term expansion for mechanistic studies. C_LIO_LI3D Esophagoid Model: A suspension-based 3D organoid system was optimized using a 96-well format, enabling high-throughput generation of esophagoids with robust epithelial stratification and mesenchymal compartmentalization. These organoids recapitulate key features of the human esophagus, including basal-to-luminal organization, and require stromal interactions for formation. C_LIO_LIFunctional Role of WNT2B in esophagus development: Both in vivo and in vitro analyses demonstrated that WNT2B regulates epithelial progenitor dynamics and tissue architecture by repressing self-renewal of basally localized TP63+ cells and inhibiting proliferation. Loss-of-function models and WNT pathway modulation confirmed its role in epithelial-mesenchymal crosstalk and organoid integrity. C_LI

Brown adipocytes (BAs) hold therapeutic promise for obesity and metabolic diseases. While interscapular BAs derive from Pax3+/Myf5+ dermomyotome, peri-aortic BAs are inferred from an unknown Pax3+/Myf5- somitic origin. Here, we identify human endotome as an MYF5-independent source of peri-aortic BAs. Through interrogating public mouse organogenesis and in-house human trunk embryoid single-cell data, we show that the early endotome cells are MYF5-independent and are primed by TGF-{beta}-induced epithelial-to-mesenchymal transition. Mechanistically, endotome-to-BA specification requires sequential BMP inhibition and Wnt activation. This roadmap results in UCP1-expressing and metabolically active BAs that transcriptionally resemble in vivo peri-aortic BAT. The multipotent endotome cells also give rise to vascular smooth muscle and endothelial cells, offering a self-sufficient source for BAT vasculature. Endotome-derived BAs show accelerated differentiation, reduced heterogeneity, and sustained Wnt activity. Thus, the endotome provides a versatile platform for generating BAs and supporting vasculature, with implications for cell-based therapy and tissue engineering in metabolic disease.

The complexity of stem cell differentiation programs remains incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogeneous cell population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow. BM-MSCs form and repair bone through the evolutionarily conserved process of endochondral ossification (EO), initiated by deposition of a transient cartilage template subsequently remodeled into bone and bone marrow tissues. Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to endochondral ossification remain elusive. To overcome donor-dependent variability in chondrogenic potential that limits mechanistic studies, we here exploit OssiGel as a potent chondro-inductive extracellular matrix offering robust recapitulation of endochondral ossification by BM-MSCs. Through multi-omics profiling of OssiGel-primed BM-MSCs, we identify rapid chromatin remodeling at chondrogenic enhancer regions as a prerequisite for lineage commitment. The emergence of a chondro-progenitor population is detected as early as 3 days in vivo, and correlates with successful EO recapitulation. Mechanistically, we identify LINC02511 as a novel enhancer-associated element involved in the onset of EO. We confirm presence of LINC02511 in human skeletal atlases, and its CRISPR-mediated silencing was shown to significantly impair EO. By integrating tissue engineering with single cell multi-omics profiling, our study provides a framework for deciphering BM-MSCs fate decisions, highlighting the role of enhancers and non-coding elements as key determinants of early lineage specification. These findings advance our understanding of BM-MSCs biology and will prompt their translational exploitation in regenerative medicine.

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

ObjectiveReliable animal models of tracheal stenosis are necessary for the development and translational testing of anti-fibrotic and regenerative therapies, but existing rabbit models frequently demonstrate substantial variability in stenosis severity, which limits their translational utility. The objective of our study was to determine whether airway diameter-matched mechanical injury improves the severity and reproducibility of experimental tracheal stenosis in a rabbit model, and to evaluate whether rabbit body weight is a reliable surrogate for tracheal luminal diameter during model creation. MethodsFourteen male New Zealand White rabbits (weight range, 2.7-3.5 kg) underwent tracheal injury using steel-bristle brushes introduced through a tracheotomy. Animals were assigned to receive either airway diameter-matched injury, in which brush size was selected to closely approximate the directly measured tracheal lumen diameter, or non-matched injury, in which brush size was selected without regard to measured lumen diameter. At postoperative day 21 (POD21), the injured tracheal segment and a native uninjured segment from the same animal were harvested and compared. Stenosis degree was quantified grossly, and lamina propria-to-cartilage (LP:C) ratio was quantified histologically by three blinded reviewers. The relationship between rabbit weight and airway diameter was assessed, and inter-rater reliability was calculated using the intraclass correlation coefficient (ICC). ResultsTwelve of fourteen rabbits reached the POD21 endpoint; two were euthanized early for severe airway compromise meeting humane endpoint criteria, both with approximately 80% stenosis. Injured tracheas demonstrated significantly greater stenosis than native controls (66.0 {+/-} 13.0% vs 16.0 {+/-} 2.7%; p = 0.00012), with a corresponding increase in LP:C ratio (p = 0.031). Airway diameter-matched injury produced significantly greater stenosis than non-matched injury (74.6 {+/-} 6.1% vs 50.6 {+/-} 4.0%; p = 0.001), while LP:C ratio did not differ between injury techniques (p = 1.0). Rabbit weight did not correlate with airway diameter (r = 0.176, p = 0.515; R2 = 0.031). Inter-rater reliability was excellent for both stenosis degree (ICC = 0.989) and LP:C ratio (ICC = 0.992). ConclusionsDirect measurement and matching of injury instrument diameter to native airway diameter substantially improves both the severity and the reproducibility of stenosis in a rabbit tracheal injury model, whereas body weight is an unreliable surrogate for airway size. This optimized, standardized protocol offers a reproducible platform for future translational studies of airway fibrosis and anti-fibrotic or regenerative therapies.

Background Donation after circulatory death (DCD) is an increasingly accepted strategy to expand the adult heart donor pool, but its use in children remains limited and incompletely characterized. We compared national characteristics and post-transplant outcomes of pediatric DCD versus donation after brain death (DBD) heart transplantation. Methods We performed a retrospective cohort study of the Organ Procurement and Transplantation Network (OPTN) registry, including patients younger than 18 years who underwent primary isolated heart transplantation between January 1993 and March 2025. Recipients were stratified by donor type (DCD vs DBD). Continuous variables were compared with the Mann Whitney U test and categorical variables with the Fisher exact test. Survival was estimated by the Kaplan Meier method and compared using the log-rank test and Cox proportional hazards regression. Results Of 10,671 pediatric heart transplant recipients, 33 (approximately 0.3%) received DCD allografts. The first DCD transplant was recorded in 2004, with a marked increase in 2023 to 2024. Compared with DBD recipients, DCD recipients were more frequently infants (<1 year, 51.5% vs 28.4%) and more often had congenital heart disease (69.7% vs 47.6%; P=0.033); DCD donors were younger (median 0 vs 6 years; P=0.038) and more frequently died of anoxia (72.7% vs 37.0%; P<0.001). Donor and recipient left ventricular mass were lower in the DCD group (P<0.05), but predicted left ventricular mass matching was similar. DCD recipients had longer hospital stays (median 31.5 vs 19 days; P=0.023); rates of treated rejection, dialysis, stroke, and pacemaker implantation were comparable. Early survival did not differ (30-day, 90-day, and 1-year), and Kaplan Meier survival through 5 years was not significantly different (hazard ratio 1.17; 95% CI 0.49 to 2.81; log-rank P=0.73). More than 90% of DCD transplants were performed in four UNOS regions (11, 4, 5, and 8). Conclusions In this national analysis, pediatric DCD heart transplantation was uncommon but expanding rapidly, concentrated in a few regions, and used preferentially in infants and children with congenital heart disease. Early post-transplant outcomes were not significantly different from DBD, supporting cautious expansion of DCD as a means of enlarging the pediatric donor pool. The small number of DCD recipients and limited followup warrant confirmation in larger, longer-term studies. Keywords: pediatric heart transplantation; donation after circulatory death; donor pool; congenital heart disease; OPTN registry; organ allocation.

Vision loss compromises the quality of life of millions of people worldwide. Currently, vision-restoring therapies are lacking. Post-mortem preservation of human eyes may enable whole eye transplantation (WET) or therapeutic development. We developed an approach to cannulate the ophthalmic artery and perfuse post-mortem pig and human eyes using a custom-built device, Eye-in-Care-Box (ECaBox). Retinal vasculature imaging and reconstruction were used to assess perfusion efficacy using a supervised deep learning model. Without intervention, the retina degrades post-mortem, whereas perfusion preserves retinal structure and cell viability for up to 24-hours. Eyes were perfused within 30 minutes post-extraction, and regained light-responses persisted for up to 10 hours after death, challenging the notion that response to light ends at death. Our findings show that intact eyes can be resuscitated and preserved outside the body, supporting the ECaBox platform for WET preservation and pre-clinical therapeutic testing. Ultimately, ECaBox may bring vision-restoring treatments to patients.

Human skeletal muscle is the principal site of insulin-stimulated glucose disposal and a major mediator of exercise-induced metabolic benefits, yet human models that preserve metabolic and exercise responsiveness remain limited. We generated primary human skeletal muscle organoids from donor-derived CD56+ myoblasts using a collagen-based extracellular matrix and serum-free IGF1-guided differentiation. The organoids formed aligned contractile tissues containing oxidative and glycolytic fiber type-like myotubes, displayed enhanced mitochondrial respiration, insulin-stimulated glucose uptake, and reproducible force generation. Electrical pulse stimulation induced AMPK activation, increased glucose utilization and lactate production, and upregulated canonical exercise-responsive genes including NR4A3 and PPARGC1A. Notably, transcriptional responses to in vitro exercise overlapped with acute exercise responses observed in skeletal muscle biopsies from the same donors. The organoids further detected functional impairments of skeletal muscle performance induced by TGF-{beta}1 and metformin and increased speed generation by testosterone treatment. These findings establish a donor-specific human skeletal muscle platform that recapitulates key features of insulin action and exercise adaptation and may enable mechanistic studies of skeletal muscle metabolism, exercise responsiveness, and therapeutic interventions relevant to diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/735246v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2ea1c9org.highwire.dtl.DTLVardef@17fa8c1org.highwire.dtl.DTLVardef@2045d5org.highwire.dtl.DTLVardef@c8b059_HPS_FORMAT_FIGEXP M_FIG C_FIG Article highlightsWe generated primary human skeletal muscle organoids under serum-free IGF1-guided conditions to reproduce key metabolic and exercise-responsive features of skeletal muscle. The organoids were insulin-responsive, displayed enhanced mitochondrial function and force-generating contractility, reproduced hallmark molecular and metabolic responses to exercise, overlapping with exercise responses observed in the same donors in vivo. The organoids were suitable to detect functional alterations after treatment with endogenous hormones and cytokines and diabetes medication This platform provides a human donor-specific system for studying skeletal muscle mechanisms underlying insulin sensitivity, exercise benefits, and therapeutic responses relevant to diabetes and metabolic disease.

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.