npj Regenerative Medicine

Tendon injuries often result in fibrosis, compromising function and predisposing to re-injury. Here, we used a full-width, non-repair Achilles tendon transection model in young (3 weeks old) and adult (18-20 weeks) rats to elucidate the cellular mechanisms governing regenerative versus fibrotic healing. Functional and biomechanical analyses revealed that young tendons recovered motion and load-bearing capacity more rapidly but exhibited more fibrotic early healing. Single-nuclei RNA sequencing identified seven major cell populations within the connective tissue compartment. Adult tendons maintained a "synthetic fibroblast" population marked by upregulated ECM synthesis, reduced stress-related gene expression, whereas young tendons favored expansion of Cxcl12/Lrp6/Gas6 fibrotic fibroblasts linked to oxidative and pro-angiogenic signaling. The young group showed sustained activation of Nox4-Gas6 pathways driving a self-reinforcing fibrotic circuit. These findings define a fibroblast lineage bifurcation that dictates oxidative stress signaling as a key regulator of fibrotic remodeling, highlighting potential therapeutic targets to promote regenerative tendon repair.

Background: For the first time, pluripotent stem cell (PSC)-derived endothelial cells were administered to a human for peripheral artery disease (PAD), in August 2026. This translation is based on a large preclinical literature claiming that donor cells build blood vessels in animal models of limb ischemia, but whether it supports that claim has not been assessed systematically. Methods: In this systematic review and meta-analysis, we searched MEDLINE, Web of Science, Europe PMC, and Embase to August 2026 for studies of PSC-derived vascular cells in animal models of limb ischemia with an acellular control. Limb perfusion at each experiment's primary timepoint was pooled as Hedges' g in a three-level random-effects model with cluster-robust variance. Donor-cell fate and a seven-level ordinal of vascular contribution were graded for every study. This meta-analysis was registered on PROSPERO (CRD420261465308). Results: Sixty-eight studies in 69 reports met the criteria; 51 contributed 114 comparisons at primary timepoints. Cell therapy increased perfusion (Hedges' g 2.26, 95% CI 1.72-2.79; P=4.1x10-11; I2=87.5%; 95% prediction interval -1.47 to 5.98). No prespecified moderator survived Holm-Bonferroni adjustment, including the donor-cell fate grade (P=0.30). A donor cell was resolved in a vessel wall in 35 studies, and flow through such a vessel in 7. Small-study effects were strong (Egger t=6.25, P=9x10-8) but were not reproduced by a sample-size-based test (P=0.80); trim-and-fill moved the estimate to 1.15. On the natural scale, treated limbs recovered about twice the control perfusion (ratio of means 1.96, 95% CI 1.66-2.33). Limb preservation favored cell therapy in 11 studies with countable events (risk ratio 4.88, 95% CI 2.28-10.45), but 43 of 68 studies reported no functional outcome, and only 22 comparisons from 8 studies were both randomized and blindly assessed. In a post hoc analysis that did not survive multiplicity adjustment, effects were largest in studies whose images could not resolve their incorporation claims (g 3.23) versus those that resolved incorporation (g 2.33). Conclusions: PSC-derived vascular cell therapy improved limb perfusion in animal models, consistently across every sensitivity analysis, although the magnitude is uncertain given heterogeneity and small-study effects. Benefit was not associated with the degree of donor-cell incorporation across studies and was largest where incorporation was least resolvable, a pattern more consistent with reporting and measurement bias than with a true mechanistic effect. The first rigorous, blinded test of that benefit may come not from animals but from the patients now receiving these cells.

Heart regeneration varies among vertebrates, with zebrafish serving as a reference species for efficient cardiac restoration. How this capacity diversified across teleosts is an emerging question, particularly following the recent identification of non-regenerative cardiac repair in medaka and cavefish. Here, we investigate heart restorative capacity following cryoinjury in two livebearers, platyfish and swordtails (Xiphophorus species), belonging to the Poeciliidae family. We demonstrate that their hearts lack the vascularized compact myocardium, a ventricular layer implicated in the restorative response in zebrafish. Following cryoinjury, both poeciliids failed to rapidly deposit fibrotic tissue that normally reinforces the damaged ventricle. This deficiency correlates with pronounced wound protrusion. Although the remaining myocardium displayed an initial proliferative response, subsequently deposited collagenous scar tissue permanently sealed the ventricular wall, precluding complete regeneration. Transcriptomic analysis identified several divergently regulated pathways between cryoinjured hearts of zebrafish and platyfish, most notably in immune response regulation. These differences were validated by delayed leukocyte infiltration and sustained inflammation in platyfish, contrasting with the rapid and self-resolving inflammatory response in zebrafish. Our findings demonstrate that Xiphophorus species have evolved hearts with compromised regenerative capacity, characterized by initial wound protrusion and permanent scarring. These results establish that lineage-specific evolutionary traits can profoundly shape regenerative competence across teleosts, with broad implications for understanding the mechanistic basis of cardiac repair. Highlights{middle dot} Viviparous poeciliids lack vascularized compact myocardium. {middle dot} Inflammation and fibrosis are delayed in the cryoinjured platyfish ventricle. {middle dot} Ventricular cryoinjury in Xiphophorus leads to transient bulging-type deformation. {middle dot} Failure to form a myocardial bridge results in permanent scarring.

Autologous nerve grafts remain the gold standard for peripheral nerve repair, but donor scarcity, donor-site morbidity and incomplete functional recovery limit their use. Schwann cells (SCs) drive autograft-mediated regeneration, yet their limited availability and expansion impede SC-based therapies. Here, we generated human pluripotent stem cell-derived SCs, motor neuron-enriched spinal cord organoids and nociceptive sensory neuron-enriched dorsal root ganglion organoids, and assembled them into a nerve-like construct termed the Peri-nervoid. Single-cell RNA sequencing revealed context-dependent SC plasticity: axonal co-culture promoted myelination, whereas axonal transection induced a repair phenotype resembling SC dedifferentiation during Wallerian degeneration. Following transplantation into 7-mm sciatic nerve defects in NSG mice, human SCs survived and remyelinated regenerating host axons, while axon-containing grafts enhanced regeneration relative to SC-only grafts. No abnormal proliferation, systemic toxicity or major-organ histopathology was detected. These findings position the Peri-nervoid as a scalable, developmentally inspired platform for developing engineered grafts for peripheral nerve repair.

Background: Zebrafish regenerate their hearts after injury, and defining the barriers that block this capacity in mammals may reveal targets for heart failure treatment. Elevated levels of the cardiomyocyte-specific kinase TNNI3K are associated with human cardiomyopathy, and its overexpression drives adverse remodeling in mice. Recent work has linked elevated TNNI3K to cardiomyocyte polyploidization and loss of regenerative competence, but whether this cell-cycle effect accounts for the pathology seen in patients has not been established. Because TNNI3K is restricted to cardiomyocytes, whether it also acts non-cell-autonomously is unknown. Methods: We generated an allelic series of zebrafish lines comprising cardiomyocyte-specific Tnni3k overexpression, an expression-matched kinase-dead variant, a full-locus deletion, and a line overexpressing mouse Tnni3k. Regeneration, cardiomyocyte proliferation, and ploidy were assessed in uninjured hearts and after cryoinjury. We combined ventricular RNA-sequencing, macrophage depletion before or after injury, tamoxifen-inducible Cre-lox switches, and cardiomyocyte-restricted CRISPR mutagenesis. Hearts from mice overexpressing human TNNI3K were analyzed in parallel. Results: Elevated Tnni3k impaired heart regeneration in a kinase-dependent manner. Cardiomyocyte proliferation, redifferentiation, and productive cell division were preserved, and polyploidization remained well below the threshold compatible with robust regeneration, arguing against a cell-cycle defect. Instead, Tnni3k overexpression established an inflammatory and fibrotic state in the uninjured myocardium, marked by interferon, NF-{kappa}B, and antigen-presentation programs and by leukocyte and macrophage accumulation. This state amplified after injury, and these animals retained substantially more scar at 60 days post-injury. Depleting macrophages before injury, but not after, prevented the excess fibrosis. Switching Tnni3k off after injury attenuated both inflammation and fibrosis. Cardiomyocyte-specific deletion of pkmb, a glycolytic gene strongly repressed in Tnni3k-overexpressing animals and previously associated with inflammation, reproduced both phenotypes. Mice overexpressing TNNI3K showed comparable fibrosis and macrophage accumulation. Conclusions: Elevated Tnni3k impairs cardiac regeneration by sustaining a chronic inflammatory and fibrotic state rather than by driving polyploidization. Because this state requires continued kinase activity and remains reversible after injury, TNNI3K inhibition may warrant exploration as a strategy to interrupt the inflammation-fibrosis loop in inflammation-driven cardiac disease.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by irreversible destruction of the alveolar epithelium and impaired regeneration. Although current therapies slow disease progression, they do not restore functional alveoli, highlighting the need for regenerative approaches that promote endogenous lung repair. Here, we performed the first unbiased functional screen of 2,042 human microRNA mimics in primary mouse alveolar type II (ATII) cells to identify regulators of ATII-to-alveolar type I (ATI) cell transdifferentiation. The screen identified miR-124-3p as the most effective promoter of ATI differentiation. In vitro, miR-124-3p promoted ATII-to-ATI transdifferentiation in healthy and bleomycin-injured ATII cells while also increasing the ATII cell pool, consistent with activity on epithelial progenitors. Using the engineered AAV6.2FF capsid, we generated a vector encoding miR-124-3p, which efficiently transduced ATII cells, MHC-II club distal progenitor cells, and injury-induced KRT8 epithelial intermediates. Therapeutic administration after fibrosis establishment reduced lung fibrosis, restored alveolar architecture, and showed greater efficacy than nintedanib in the bleomycin mouse model. Mechanistically, we propose a context-dependent model whereby miR-124-3p regulates epithelial cell states through the EZH2-C/EBP axis while attenuating epithelial transcriptional programs associated with IPF. Together, these findings support AAV-mediated delivery of miR-124 to promote alveolar repair in pulmonary fibrosis.

Neonatal mammals exhibit a remarkable capacity for cardiac repair that declines rapidly after birth and continues to diminish with age. This loss of regenerative potential is accompanied by age-dependent changes in macrophage behavior, shifting from pro-regenerative to pro-fibrotic responses following injury. We investigated whether extracellular matrix (ECM)-derived matrikines from different developmental ages regulate macrophage polarization. Cardiac ECM was isolated from neonatal (P2), adolescent (6-week), and adult (16-20-week) rat hearts through decellularization and pepsin digestion. ECM peptides were adsorbed onto tissue culture surfaces, and murine RAW 264.7 macrophages were cultured on ECM-coated or uncoated controls. After 24 hours, macrophage phenotype and gene expression were assessed using marker analysis and RNA sequencing. Macrophages cultured on neonatal cardiac ECM displayed reduced inflammatory activation and maintained expression of pro-reparative markers, even in the presence of inflammatory stimuli. Transcriptomic analysis revealed a distinct ECM-driven phenotype that differed from canonical M1 and M2 polarization states. Gene ontology analysis demonstrated significant downregulation of pathways associated with antiviral responses, immune activation, and interferon signaling in macrophages exposed to neonatal ECM. These effects were not observed with adolescent or adult ECM. Collectively, these findings suggest that neonatal ECM matrikines suppress interferon-mediated inflammatory signaling, potentially contributing to the reduced inflammation and enhanced repair observed following myocardial injury in neonatal hearts.

Chronic inflammation drives persistent systemic cytokine signaling that contributes to vascular dysfunction and secondary vasculitis, yet mechanistic studies are limited by models that fail to capture the multicellular architecture and dynamics of human arteries. In contrast, perfusing intact vessels ex vivo has limited tractability because of material availability and difficulty of genetic or biochemical manipulation. We developed a modular, perfused artery-on-a-chip platform by tri-axially bioprinting primary human vascular cells to recapitulate the concentric organization of the intimal, medial, and adventitial layers. The engineered vessels are viable longer than 21 days, with functional endothelial barriers, contractile smooth muscle behavior, and actively remodeled extracellular matrices bearing hallmarks of native vascular tissue. Addition of tumor necrosis factor alpha (TNF) induces altered transcript levels of proinflammatory mediators and secretion of cytokines and matrix-remodeling enzymes without compromising vessel viability. Importantly, this secretory response is effectively attenuated by both a small-molecule JAK1 inhibitor (ABT-317) and anti-TNF antibody (Infliximab), demonstrating the models utility for therapeutic evaluation.

Astrocyte border formation is a conserved neuroprotective response to neural tissue disruption, yet astrocyte border states at implanted biomaterials remain less well characterized than injury responses. Here, we developed the Astrocyte Border Characterization (ABC) Tool, which leverages a shear-thinning, injectable biomaterial to locally deliver astrocyte-specific RiboTag AAVs and small molecule regulators in the mouse striatum, enabling molecular profiling and phenotypic modulation of astrocyte border (AB) cells. Spatially precise delivery of AAV using the ABC Tool yielded enhanced specificity and robust RiboTag expression in AB cells from 7-70 days post injection. Temporal transcriptomic profiling of AB cells revealed predominantly acute, transient changes in genes governing dedifferentiation, proliferation, metabolic reprogramming, and inflammation regulation. Persistent changes accounted for only 14% of regulated genes but involved critical gain of functions in immune regulation and host defense that mirrored astrocyte border responses at chronic CNS injuries. Local delivery of indiscriminate or astrocyte-selective ablation molecules delayed, rather than prevented, border formation, ultimately yielding thicker astrocytes borders with increased inflammation and fibrosis at the biomaterial-tissue interface. Conversely, local delivery of {beta}-hydroxybutyrate (BHB) from the ABC Tool altered key aspects of the transcriptional reprogramming to attenuate chronic astrocyte reactivity and prevent biomaterial contraction without exacerbating inflammation or fibrosis. Our findings establish the ABC Tool as a bioassay for studying and manipulating astrocyte borders at implanted biomaterials and identify focal metabolic regulation as a strategy to modulate AB cell phenotypes and enhance the CNS biocompatibility of biomaterials.

BACKGROUND: Increased left ventricular mass (LVM) at a single time point after heart transplantation (HT) predicts future adverse outcomes. However, dynamic changes in LVM could have better biological relevance and reflect adverse graft remodeling (AGR). The prognostic significance of such serial changes has not been studied. METHODS: Using an automated, electronic health record-based institutional data infrastructure, we studied 439 HT recipients with 5,563 LVM measurements. Separate Bayesian joint models estimated the simultaneous associations of current LVM and its instantaneous rate of change with graft dysfunction (GD) and mortality. A joint-model-derived remodeling score combining patient-specific deviations in LVM and slope was dichotomized to define AGR and non-AGR groups. A mixed-effects analysis of all clinical variables was performed to assess associations with LVM both between and within patients. An independent cohort of 35 patients with 79 surveillance-biopsy RNA-sequencing samples was used to examine early stress-responsive pathways associated with the remodeling score. RESULTS: LVM declined by approximately 7 g/year after transplantation, with regression attenuating over time. Sixty patients (13.7%) had GD, and 75 (17.1%) died. Higher LVM was associated with subsequent GD (hazard ratio [HR] per 10 g, 1.14; 95% credible interval [CrI], 1.02-1.28) and mortality (HR, 1.10; 95% CrI, 1.02-1.19). A more positive LVM slope was associated with GD (HR per 1 g/year, 1.21; 95% CrI, 1.06-1.42) and with cardiac allograft vasculopathy (CAV) grade 2 or 3 (HR, 1.39; 95% Crl, 1.02-1.96). LVM regressed more slowly in the AGR group (-5.8 vs -8.4 g/year), with higher GD (21.0% vs 6.4%) and mortality (24.2% vs 10.0%). Time-updated GD was associated with subsequent death (HR, 8.12; 95% Confidence Interval [CI], 4.67-14.14). Transcriptomic analysis showed enrichment of interferon-mediated signaling and vascular endothelial activation with higher remodeling scores, whereas lower scores were associated with mitochondrial and metabolic processes, ribosome biogenesis, and pathways related to tissue repair and stress responses. CONCLUSIONS: AGR is an easily accessible imaging biomarker that reflects the changes in the allograft in response to various stressors and predicts future adverse outcomes. Discovery of molecular mechanisms of AGR could lead to novel therapies to protect the allograft from chronic rejection.

The ability to generate vascularized tissue constructs supplied by surgically accessible vessels remains a major limitation in reconstructive tissue engineering. Previous work demonstrated that positioning porous biomaterials around a vein can induce extensive vascular sprouting and generate a vascularized tissue volume without creation of an arteriovenous loop. However, this phenomenon has principally been demonstrated using rigid calcium-phosphate scaffolds, limiting its direct relevance to soft-tissue reconstruction. Here, we investigated whether a flexible decellularized dermal matrix could similarly be vascularized around a single unmodified vein and whether autologous dermal fibroblasts could further enhance this response. Decellularized rat dermis, with or without autologous dermal fibroblast seeding, was implanted either subcutaneously in the dorsal region or around the femoral vein for 8 weeks. Following Microfil perfusion, three-dimensional micro-CT demonstrated an extensive branching vascular network within perivenous constructs. Vessel volume relative to tissue volume was 0.2 +/- 0.1% in subcutaneous constructs without fibroblasts (n = 11) and 0.6 +/- 0.2% with fibroblasts (n = 9), compared with 6.+/- 1.4 % in perivenous constructs without fibroblasts (n = 10) and 7.3+/- 1.2% with fibroblasts (n = 10). Histology and immunohistochemistry similarly showed greater cellular repopulation and substantially higher CD31- and -SMA-positive vessel densities following perivenous implantation. Integrated two-way ANOVA of the same 8-week data confirmed a dominant effect of implantation site across the vascular outcomes, while fibroblast seeding significantly affected vascular volume, dermal remodeling, and nuclear density. The effect of fibroblast seeding on vascular volume was site dependent, with a significant implantation site fibroblast interaction. At 8 weeks, the vascularized dermal construct was completely dissected free from the surrounding tissues while maintaining its connection to the femoral vein. A small superficial incision into the isolated dermis produced visible bleeding, supporting an acute functional connection of the intradermal vascular network to the host circulation while the femoral venous connection was preserved. After flap elevation, perivenous constructs retained substantially greater vascular volume and showed more complete surface healing and organized skin architecture than subcutaneous constructs. Fibroblast seeding accelerated wound closure, while its effect on post-elevation vascular volume depended on implantation site: VV/TV decreased in subcutaneous constructs but increased in perivenous constructs. These findings show that single-vein vascularization can extend from rigid calcium-phosphate biomaterials to a compliant biological extracellular-matrix scaffold and may provide a simplified axial vascularization strategy without arterialization or incorporation of an arterial vessel.

The alveolar sac architecture is essential for efficient gas exchange and must be precisely maintained throughout life; however, how this delicate structure is preserved during adult regeneration remains poorly understood. Using a mouse pneumonectomy model, we found that Lgr6+ Hhip+ ductal myofibroblasts, a poorly characterized mesenchymal population, are indispensable for maintaining alveolar integrity during lung regrowth. Comprehensive characterization using single-cell transcriptomics, mouse genetics, and pharmacological assays demonstrated that these ductal myofibroblasts secrete myogenic factors, most notably CCN4, to reactivate a myogenic program that converts them into contractile PA-DMFs, thereby preserving alveolar architecture. Lineage-tracing further revealed that these ductal myofibroblasts originate from embryonic MCAM- SMA+ distal progenitors via subepithelial TGF-{beta} signaling, serving as a lifelong guardian of alveolar structural integrity. Notably, cross-species analysis identified an analogous population of LGR6+ fibromyocytes in human respiratory bronchioles. Together, these findings indicate ductal myofibroblasts as a developmentally programmed cell population that reactivate a contractile program to structurally support the regeneration of adult lungs.

In the US, 33 million musculoskeletal injuries have been reported per year, with 50% involving tendons and ligaments in both athletic and aging populations. Tendon repair often results in the formation of biomechanically inferior scar tissue rather than functional regeneration. Local cell therapy is garnering significant interest in tendon repair because it provides a targeted means to repopulate defects with potent therapeutic cells. Here, we developed a porcine tendon-derived thermoresponsive extracellular matrix hydrogel (TG) as an injectable, bio-instructive carrier for scleraxis-overexpressing induced mesenchymal stem cell derived tenocytes (iTenocytes), with the goal of improving cell retention and overall transplantation success. TG was compositionally distinct from purified collagen (PC, from rat tail type 1 collagen) and exhibited favorable material properties for a minimally invasive percutaneous strategy, including thermoresponsive gelation, shear-thinning injectability, retention at the injection site, and controlled biodegradation. In vitro, TG supported three-dimensional cell residence, promoted cell interconnectivity and redistribution at the matrix interface, and increased collagen type I release from iTenocytes compared to those embedded in PC. Transcriptomic and proteomic analyses further showed that TG enhanced programs associated with tenogenic maturation, extracellular matrix assembly, focal adhesion, mechano-transduction, and remodeling. In a rat Achilles tendon partial defect model, both optical imaging and histological analyses demonstrated retention of iTenocytes at the injection site. Additionally, confocal imaging demonstrated retention of iTenocytes within the defect site for up to 10 days. Together, these findings identify TG as a biofunctional injectable carrier that supports the tenogenic characteristics of iTenocytes and enhances their local persistence after transplantation.

Introduction: Placental tissue allografts are widely utilized in the management of chronic wounds, including diabetic foot ulcers. In addition to serving as wound coverings, these products may influence wound repair through extracellular matrix architecture and biologically active signaling molecules native to human amniotic tissue. Manufacturing and processing methodologies vary considerably across commercially available products and may substantially impact biologic activity, tissue remodeling, and wound-healing trajectories. In this study, a rodent wound-healing model was utilized to compare a proprietary processed placental tissue allograft with a conventionally processed placental tissue comparator. Methods: Two arms of a five-arm pilot rodent wound-healing study were evaluated to compare a proprietary processed human amniotic tissue allograft against a conventionally processed comparator; the remaining arms evaluated test articles under development and will be reported separately. Male rats (n=8 per arm), matched for weight, age, and diet, each received two full-thickness dorsal excisional wounds approximately 10 mm in diameter and 2 mm deep. Test articles were reapplied upon resorption over a 14-day period. At Day 14, sections were scored semi-quantitatively (0-4 Severity Injury Score) across histologic and immunohistochemical parameters spanning epidermal repair, inflammation, angiogenesis, proliferation, and remodeling. Cranial and caudal wounds were averaged within each animal, retaining the animal as the experimental unit. A Day 14 Histology Resolution Score was derived as a signed composite of pro- and anti-regenerative markers and evaluated alongside percent wound closure. Inferential statistics (Welch ANOVA with Games-Howell post hoc) were derived from the full five-arm cohort (n=40). Results: Animals treated with the proprietary processed allograft showed more advanced progression toward wound resolution than those receiving the conventionally processed comparator, across both surface and tissue-level endpoints. Mean animal-level wound closure at Day 14 was 95.2% with the proprietary processed allograft versus 89.9% with the conventional comparator. The integrated Day 14 Histology Resolution Score, a composite of epidermal repair, inflammation, angiogenesis, proliferation, and remodeling endpoints was 8.94 versus 5.13, corresponding to a shift from the poor-resolution range into the partial-resolution range on the pre-defined interpretive scale. Welch ANOVA across the full five-arm cohort (n=40) detected a significant overall effect of treatment on this composite (p = 0.010). The pairwise contrast between these two arms did not reach significance under Games-Howell correction across ten comparisons (p = 0.337); at n=8 per arm the study was not powered to resolve individual pairwise differences, and the observed standardized difference was large (Hedges' g = 0.93). Injury endpoints were consistently lower in the proprietary processed allograft: 69% lower acute inflammation, 60% lower dermal necrosis, approximately 57% lower epithelial erosion, 46% lower ulceration, and 42% less hemorrhage. Importantly, these reductions in residual tissue injury occurred while maintaining robust granulation tissue formation, angiogenesis, collagen remodeling, cellular proliferation, and macrophage recruitment. Discussion: These findings suggest that processing methodologies can materially influence the biologic performance of placental tissue allografts. The proprietary processed allograft was associated with lower residual tissue injury and greater regenerative and reparative activity relative to a conventionally processed comparator. These observations support the concept that placental tissue allografts should not necessarily be viewed as biologically equivalent and may have important implications for clinical outcomes and product selection in wound care.

Living myocardial slices (LMS) are a highly relevant ex vivo model for investigating cardiac physiology and disease, as they preserve the native three-dimensional architecture, cellular diversity, and extracellular matrix of the heart. In addition, LMS enable longitudinal functional and molecular analyses. In this study, we established and compared two LMS culture approaches: an air-liquid interface system and a biomimetic culture system. We further examined how different slicing techniques affect tissue quality and longevity within the biomimetic setup. To develop a fibrosis model, LMS were stimulated with transforming growth factor-beta1 (TGF-beta1) and/or exposed to increased mechanical load. Tissue viability was assessed using LIVE/DEAD staining and the MTT assay, while cytotoxicity was evaluated with the LDH-Glo-TM Cytotoxicity assay. Contractile function was measured, and fibrotic remodelling was analysed using RT-qPCR, ELISA, and immunohistochemistry. Our results demonstrate that LMS cultured in the biomimetic system exhibit superior viability, structural integrity, and functional performance compared with those maintained at the air-liquid interface. Mouse LMS could be stably cultured for up to one week in the biomimetic system. Importantly, sample preparation, particularly the slicing method, had a significant impact on tissue quality and culture duration. While TGF-beta1 stimulation alone did not consistently induce fibrosis, combining TGF-beta1 treatment with increased mechanical load led to more pronounced fibrotic remodelling in LMS. These findings highlight the importance of biomechanical cues in modelling cardiac fibrosis ex vivo and support the biomimetic system as a robust platform for functional and disease-relevant studies.

Decellularized extracellular matrix (dECM) scaffolds are increasingly used in regenerative medicine, yet the extent to which processed placental dECM retains properties capable of influencing cellular responses remains unclear. This study combines functional cell assays with deep learning-enabled quantitative imaging to determine how dehydrated placental ECM regulates cellular behavior across multiple human cell lineages. Human dermal fibroblasts, cardiac fibroblasts, and osteoblasts were cultured on dehydrated placental ECM or standard cell culture surfaces and assessed for cell attachment, viability, extracellular matrix production, and nuclear morphology. Placental dECM supported attachment and survival across all three cell types, while Pro-Collagen I Alpha 1 secretion varied by cell lineage relative to negative controls. To identify structural responses associated with scaffold culture, an automated imaging pipeline combining Cellpose-based nuclear segmentation with nuclear morphometric analysis was used to quantify nuclear area, eccentricity, and circularity. Quantitative profiling of hundreds of nuclei revealed scaffold-dependent remodeling of nuclear morphology that was not apparent by conventional microscopy. Cells cultured on placental dECM exhibited reduced nuclear area and increased nuclear eccentricity, while cardiac fibroblasts and osteoblasts showed alterations in nuclear circularity. These lineage-dependent morphological responses demonstrate that placental dECM provides more than a permissive substrate for cell attachment and is associated with measurable changes in cellular architecture following processing. Together, these findings support the biological relevance of processed placental dECM as a regenerative biomaterial and demonstrate the utility of quantitative single-cell morphometric analysis for detecting cell-material interactions that may not be apparent through qualitative imaging alone, guiding the rational design of regenerative therapies.

Background Hypertrophic and dilated cardiomyopathies (HCM and DCM) are the most common inherited cardiomyopathies. However, genotype-specific molecular and functional cardiomyocyte phenotypes and responses to neurohormonal stimulation remain incompletely understood. Here, we investigated whether patient-derived HCM and DCM cardiomyocytes exhibit distinct baseline phenotypes or differential responses to hypertrophic stimulation and pharmacological treatment. Methods Three human-induced pluripotent stem cell (hiPSC) lines were used: a control line, an HCM patient-derived line carrying a MYBPC3 mutation, and a DCM patient-derived line carrying an LMNA mutation. The cells were differentiated into hiPSC-cardiomyocytes, which were exposed to endothelin-1 and the GATA4-targeted compound 3i-1262, followed by transcriptional and protein expression analyses. In addition, engineered heart tissues (EHTs) were generated and cultured for 40 days, with endothelin-1 and 3i-1262 treatment applied during the final 20 days. Lastly, {beta}-adrenergic stimulation with isoprenaline was performed. EHT contractile function was quantified using MUSCLEMOTION. Results Patient-derived hiPSC-cardiomyocytes exhibited genotype-dependent responses to endothelin-1 at the transcriptional and protein levels. DCM-cardiomyocytes failed to maintain structural integrity in the EHTs, resulting in tissue fracture or cessation of beating. Functional analyses demonstrated distinct baseline contractile properties between control and cardiomyopathy EHTs, as well as differential responses to endothelin-1 and isoprenaline. Conclusions Patient-derived hiPSC-cardiomyocytes exhibit genotype-specific molecular and functional phenotypes. EHTs generated from HCM hiPSC-derived cardiomyocytes showed a progressive decline in apparent force, whereas DCM EHTs fractured over time, suggesting mutation-associated phenotypes in 3D cardiac tissue models. These findings highlight the utility of hiPSC-based cardiac models for investigating molecular and functional disease mechanisms and pharmacological responses in inherited cardiomyopathies. Keywords: hypertrophic cardiomyopathy, dilated cardiomyopathy, engineered heart tissues, hiPSC-derived cardiomyocytes, GATA4

Transplantation is limited by how long organs survive outside the body. Existing preservation methods slow metabolism or sustain physiological support, but neither eliminates ischemia-reperfusion injury (IRI), which shortens storage and drives organ discard. Embryonic diapause and hibernation show that mammalian cells retain a latent capacity for reversible metabolic arrest. Screening the PI3K-mTOR axis, we identified a dual inhibitor that pauses embryonic stem cells reversibly at ambient oxygen and holds intact mouse embryos in arrest for 11 days. Combined with a mitochondria-targeted hydrogen sulfide donor, this yields TorPause: a two-component flush pairing metabolic pausing with mitochondrial protection, compatible with existing preservation protocols. In a rat liver transplantation model, TorPause reduced structural injury and improved recipient survival after severe warm ischemic storage, potentially expanding the usable donor pool.

Fibrotic skin diseases are characterized by persistent fibroblast activation and extracellular matrix remodeling, yet the mechanisms governing fibroblast state transitions remain incompletely understood. Here, we established a human iPSC-derived skin organoid model of fibrosis through chronic TGF-{beta} stimulation. Single-cell RNA sequencing combined with immunofluorescence-based spatial analysis revealed dynamic fibroblast state transitions, spatial reorganization, and expansion of activated fibroblast populations during fibrotic remodeling. Integration with human scleroderma single-cell datasets demonstrated conserved fibroblast states and transcriptional programs between organoids and patient tissues. We further identified broad induction of RUNX2 in the dermal compartment during fibrosis, and RUNX2 depletion attenuated fibrotic marker expression. CUT&RUN profiling revealed RUNX2 occupancy at fibrosis-associated loci, including RUNX1 and LOXL2. Using a machine learning-guided screening approach, we identified F0565-0303, a small molecule that suppressed RUNX2-dependent fibrotic programs in vitro and reduced fibrosis in a bleomycin-induced mouse model. Together, these findings establish human skin organoids as a platform for modeling fibrosis and nominate RUNX2 as a potential therapeutic target.

Retinal organoids (ROs) model human retinogenesis but remain avascular and lack immune components, limiting their utility for studying vascular retinopathies. We generated vascular organoids (VOrs) from human pluripotent stem cells and evaluated their integration into developing ROs. Dissociated VOr cells (day 12 or day 30) were seeded onto 2D retinal neuroepithelium before 3D transition, followed by long-term culture to day 210. VOr derived cells migrated into ROs and transiently persisted. V30 ROs preserved retinal lamination, major neuronal subtypes and light evoked retinal ganglion cell responses comparable to non vascularised controls, whereas media-only ROs cultured in proangiogenic conditions showed structural disorganisation and reduced neuronal survival. Single-cell RNA sequencing revealed an endogenous, inflammatory endothelial-like population with partial endothelial-to-mesenchymal features and a perivascular fibroblast-like cluster in pro-angiogenic media only RO controls. The endothelial-like cell cluster was significantly enriched for genes upregulated in human retinal endothelial cells exposed to high glucose, indicating a diabetic retinopathy like transcriptional state. Microglia like and border associated macrophage populations were present across all conditions, with developmental shifts in morphology and marker expression. Our findings define conditions under which vascular cells can be incorporated into human ROs without disrupting retinal architecture, while uncovering an intrinsic capacity for inflammatory endothelial activation that partially recapitulates diabetic retinopathy. This work advances the engineering of vascularised, immunocompetent retinal models and highlights endogenous vascular immune programmes that must be considered when modelling retinal disease in vitro.