npj Regenerative Medicine

Extracellular matrix (ECM) scaffolds induce type 2 immunity to promote repair. Here, we show that immune cells recruited to ECM-treated murine muscle injuries and clinical soft tissue defects express immune checkpoints. Specifically, TH2 cells and regulatory T cells (Tregs) increase LAG3 expression, while macrophages express PDL2. TCR analysis and a triple-reporter strain for interleukin (IL)-13 and Treg fate-mapping suggest that Tregs in ECM-treated wounds transition into TH2-like exTregs that express LAG3. Immune checkpoint inhibition (ICI) significantly stimulated type 2 immunity in ECM-treated wounds, including increased TH2 cells, Treg transition to TH2-like exTregs, and pro-regenerative macrophages. Moreover, ICI enhanced muscle repair and reduced fibrosis in ECM-treated wounds. Collectively, these findings show Treg/TH2 plasticity in wound healing and introduce a novel ICI application to enhance immune-mediated regeneration.

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

Macrophages play a central role in early immune response after injury that can shape the success or failure of craniomaxillofacial (CMF) bone repair. While mineralized collagen glycosaminoglycan (GAG) scaffolds have been developed to support osteogenesis, here we define how scaffold pore size, pore alignment, and glycosaminoglycan (GAG) composition influence human monocyte-derived macrophage polarization. We establish flow cytometry, secretome, and gene expression benchmarks to assess primary macrophage polarization toward M1 versus M2 phenotypes in response to cytokine cocktails in 2D culture and 3D scaffolds. We then define the kinetics macrophage polarization in response to scaffold pore architecture and composition in the absence of exogenous cytokines. All scaffold variants support an early pro-inflammatory response followed by a shift toward M2-like phenotypes over seven days reflected by increased CD206 expression, secretion of pro-healing factors such as CCL18, and upregulation of M2a- and M2c-associated genes. Anisotropic scaffolds with smaller pores more robustly drove angiogenic and extracellular matrix related gene expression as well as earlier emergence of M2-like phenotypes. Scaffold GAG chemistry provided an additional tuning mechanism, with chondroitin-6-sulfate variants promoting the greatest late-stage M2 surface marker expression, heparin variants accelerating early M2 and pro-angiogenic phenotypes, and chondroitin-4-sulfate variants dampening both M1 and M2 phenotypes at early timepoints. These findings demonstrate that mineralized collagen scaffolds intrinsically guide macrophage polarization toward pro-regenerative states but that scaffold structure and composition can be used to shape the kinetics and intensity of these responses. These insights provide a critical foundation for immuno-instructive biomaterial designs that enhance CMF bone repair.

Despite the growing prevalence of non-healing diabetic wounds, no current treatment options overcome multifactorial deficits in repair. To this end, a mesenchymal stromal cell-derived regenerative extracellular matrix (rECM) was evaluated for the ability to accelerate cutaneous wound repair in leptin receptor-deficient (db/db) diabetic mice with paired full-thickness dorsal skin defects. A single dose of rECM significantly accelerated wound closure compared with vehicle controls. Also, rECM dose-dependently improved overall histological healing scores and modulated granulation tissue dynamics, with the highest dose promoting rapid resolution of granulation tissue relative to wound area. Spatial transcriptomics and immunofluorescence revealed that rECM drove robust formation of de novo peripheral nerve clusters characterized by the Schwann cell marker, p75. The rECM also enhanced vascular maturation in healed wounds, increasing average blood vessel size, smooth muscle actin-positive vessels, and vessel density within myofibroblast-rich regions. In a complementary 3D angiogenic sprouting model, rECM accelerated endothelial invasion and filopodia extension, and at higher concentrations induced contraction of collagen matrices consistent with accelerated resolution of granulation tissue. These data demonstrate that rECM accelerates closure of diabetic skin defects by coordinating faster granulation tissue remodeling with enhanced peripheral nerve formation and vascular maturation.

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

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

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

Volumetric muscle loss (VML) injuries overwhelm the inherent regenerative capacity of skeletal muscle, causing persistent functional deficits with no routinely effective therapies. Electrical stimulation (ES) has been shown to preserve muscle structure in other injury models, but technical barriers have prevented daily delivery during the acute post-injury window when critical regenerative programs are established. Here, we developed a fully implantable bioelectronic system with nanoporous platinum-modified electrodes enabling daily therapeutic stimulation and electromyographic recording without repeated anesthesia in a rat tibialis anterior VML model. Animals receiving ES during the acute post-injury period (10 sessions over days 0-14) showed sustained functional improvement, reaching 86.5% of baseline torque at 8 weeks compared to 68.1% in unstimulated controls. This recovery reflected enhanced remodeling of injured muscle rather than synergistic muscle compensation. Histological analysis revealed coordinated early increases in vascularization, pro-regenerative macrophages, and satellite cells. These findings establish early ES as a promising intervention for promoting muscle regeneration after catastrophic injury.

The rotator cuff is a group of four muscles in the shoulder, which aid in movement and rotation of the upper arm. Rotator cuff tears (RCTs) within tendons of these muscles are common musculoskeletal injuries, often resulting in intramuscular fat, fibrosis, and muscle atrophy. Fatty infiltration specifically correlates with high rates of retear following repair. The cellular sources and molecular cues that cause these pathologies are unknown and therefore non-surgical cell/drug therapies for RCTs do not exist. Thus, we first sought to determine the cellular source(s) and molecular underpinnings of fatty atrophy and fibrosis associated with massive RCTs. Using a murine model of massive RCTs combined with lineage tracing, we demonstrate that muscle resident Pdgfra+ non-myogenic mesenchymal cells (NMMCs) are responsible for the fatty and fibrotic RCT pathologies. Utilizing sorted Pdgfra+ cells from rotator cuff muscles and "deep" single cell RNA-sequencing, we identified a specific Dpp4+ cell population associated with RCT-induced fibrosis, while Gfra1+ nerve-associated NMMCs are drivers of the RCT-induced intramuscular fat pathology. Finally, we demonstrate that RCT-induced fatty infiltration occurs at least partially via the loss of GDNF-GFRA1-RET signaling, since local treatment of murine RCTs with a small molecule RET agonist reduces development of the RCT-induced intramuscular fat.

Chronic diabetic wounds represent a major clinical burden and are strongly associated with peripheral neuropathy, yet the contribution of nerve-associated Schwann cells to impaired healing remains poorly defined. Here, we investigated Schwann cell dynamics in cutaneous wound repair using the db/db model of type 2 diabetes. Full-thickness excisional wounds in db/db mice exhibited delayed closure, reduced dermal and epidermal thickness, and diminished cellular proliferation compared to non-diabetic controls. Diabetic wounds also demonstrated impaired re-innervation and a marked reduction in both total (S100{beta}+) and dedifferentiated (p75NTR+) Schwann cells, including decreased Schwann cell proliferation. These findings indicate that diabetes disrupts the injury-induced Schwann cell response that is essential for normal repair. Transcriptomic analyses revealed that injury-activated Schwann cells upregulate multiple trophic factors, including oncostatin M (OSM), while single-cell RNA sequencing demonstrated broad expression of OSM receptors (Osmr and Il6st) across wound-resident keratinocytes, fibroblasts, and vascular-associated cells, suggesting widespread responsiveness to OSM signalling during repair. Therapeutic administration of OSM to diabetic wounds significantly accelerated closure, reduced wound width and area, and increased dermal and epidermal thickness. Mechanistically, OSM enhanced epidermal proliferation, angiogenesis, and cutaneous axon regeneration. Collectively, these data identify Schwann cell dysfunction as a contributor to impaired diabetic wound healing and demonstrate that augmenting a Schwann cell-derived paracrine signal can partially rescue key reparative processes. Our findings support a regulatory role for Schwann cells in coordinating epithelial, vascular, and neural repair responses and highlight OSM signalling as a potential therapeutic strategy for chronic diabetic wounds.

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

Peritendinous adhesions are a debilitating complication of tendon injury characterized by excessive matrix deposition and chronic inflammation. Due to limitations of current preclinical models, the underlying mechanisms of adhesion pathogenesis remain poorly defined, and there are no approved drugs to prevent or resolve adhesions. Here, we develop a human synovial tendon-on-a-chip (synToC) that integrates synovial fibroblasts, tendon-resident fibroblasts, immune cells, and vascular endothelium to reconstruct the intrasynovial tendon microenvironment. We show that synovial fibroblast activation promoted tendon contraction and inflammatory cytokine secretion dominated by IL-6, leading to monocyte infiltration and formation of fibronectin- and collagen III-rich matrix bridges between tendon and synovial compartments resembling nascent peritendinous adhesions. These phenotypes emerged even in the absence of exogenous TGF-{beta}1, indicating that synovial fibroblast-mediated crosstalk is sufficient to initiate adhesion-like pathology. Importantly, pharmacological inhibition of the IL-6/JAK/STAT pathway suppressed synovial activation, blunted inflammatory cytokine signaling, and attenuated fibrotic matrix deposition and interfacial adhesion formation. These findings establish the synToC as a human-relevant new approach methodology (NAM) to interrogate the multicellular drivers of tendon adhesions and to accelerate the development of anti-fibrotic therapies.

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

Acellular Adipose Tissue (AAT) is an off-the-shelf, cadaveric adipose-derived ECM-based biomaterial for soft tissue reconstruction. AAT has been validated preclinically to promote angiogenesis and adipogenesis and demonstrated safety, biocompatibility, and tolerability in a Phase I study. In this study we report the findings for the first ten patients in the Phase II study for permanent reconstruction of modest soft tissue defects. AAT promoted macrophages, CD3+ T cells, and CD34+ progenitor activity. Multiplex immunofluorescence staining using the PhenoCycler (formerly CODEX) imaging platform found that AAT can induce tertiary lymphoid structures (TLS). Nanostring GEOMx spatial transcriptional data analysis found significant differential gene expression between neighboring tissues with EGR1, MCL1, and NR4A1 upregulated in AAT. These genes have roles in angiogenesis, anti-apoptotic processes, and promotion of anti-inflammatory genes, respectively. AAT promoted anti-fibrotic CD74+ adipose-derived stromal cells, confirmed by immunofluorescence staining. Our findings demonstrate that AAT promotes angiogenesis, adipogenesis, and anti-fibrotic remodeling.

The long-term performance of tissue-engineered scaffolds, particularly small-diameter vascular grafts, is shaped by remodeling events at the tissue-graft interface, yet these processes remain difficult to resolve longitudinally and at microstructural resolution in conventional implantation models. Here we develop an organotypic artery-graft model that preserves cylindrical vessel geometry and enables non-destructive label-free multiphoton monitoring of interface remodeling. Using second harmonic generation and two-photon excited fluorescence, we capture evolving fibrillar collagen architecture and cellularization over time, demonstrate compatibility with multiple biomaterial classes, and show integration with rat and mouse explants, live-cell dyes, and fluorescent reporter tissues. The platform resolved distinct remodeling responses to transforming growth factor-{beta} isoforms (TGF-{beta}1, -{beta}2, and -{beta}3), with differential shifts in collagen-fiber distributions, accompanied by changes in matrix-remodeling and contractile gene expression. Across two graft designs, culture-derived remodeling phenotypes, collagen fiber distributions, and initial trajectories agreed with those observed in long-term 6-month interpositional explants. Together, these results establish an accessible intermediate platform for interrogating artery-graft remodeling, tracking these trajectories, and prioritizing graft designs through interface-resolved outcomes before and alongside animal implantation studies.

Critical sized craniomaxillofacial bone defects do not heal naturally and often exhibit chronic inflammatory responses that restrict regeneration. It is increasingly apparent that biomaterials must facilitate dynamic crosstalk between immune cells, such as macrophages, and osteoprogenitors to resolve inflammation and accelerate regeneration. Here, we evaluate interactions between macrophages in a neutral (M0) or pro-inflammatory (M1) state with mesenchymal stem cells (MSCs) in a basal or licensed state within a mineralized collagen scaffold. We reveal that MSC-macrophage crosstalk influences significant changes in osteoprogenitor cell differentiation and immune cell polarization. Notably, crosstalk between MSCs and macrophages drives an early-stage inflammatory response, which enhances the immunomodulatory activity of MSCs via secretion of IL-6, an effect that is heightened for already licensed MSCs. The presence of macrophages in the co-cultures upregulated osteogenic (ALPL, BMP2, COL1A2, and RUNX2) and angiogenic genes (ANGPT1) in basal MSC groups. Further, MSC-macrophage interactions subsequently drive increased M2-like macrophage polarization as early as 7 days of culture, as indicated by surface marker expression. These findings show that biomaterial scaffolds can be leveraged as mediators of MSC-mediated immunomodulation with an emphasis on achieving early-stage pro-inflammatory phenotypes that drive subsequent macrophage polarization and markers of increased regenerative potency.

Engineered cell therapies present an opportunity for endogenous, site-specific production of therapeutic agents. Here we describe a closed-loop cell therapy which secretes an inhibitor of Activin A, ActR2A-Fc, upon exposure to Activin A. We demonstrate in vivo therapeutic efficacy of this approach in a mouse model of fibrodysplasia ossificans progressiva (FOP), a morbid condition in which patients develop extensive heterotopic bony lesions in response to aberrant sensitivity to Activin A through a mutation in the type I BMP receptor ACVR1 (ACVR1 R206H). To blunt Activin A activity, we designed a transposon plasmid containing the transgene encoding ActR2A-Fc, with expression controlled by the BMP-responsive element (BRE). In cells containing the causative mutation, the BRE is pathologically activated upon exposure to Activin A. FOP-derived marrow cells modified with the BRE-ActR2AFc plasmid exhibited the desired closed-loop functionality, with increased ActR2A-Fc expression upon exposure to Activin A and reduced expression upon withdrawal of Activin A. Engineered marrow cells secreted bioactive ActR2A-Fc, and bone marrow transplantation of FOP marrow cells engineered with the BRE-ActR2AFc transposon into same-sex FOP mice resulted in absence of heterotopic bony lesions. Experiments with labeled, engineered FOP marrow cells verified trafficking of the therapeutic cells to sites at risk for FOP. These data provide proof-of-concept for the therapeutic utility of engineered cell therapy for the treatment of FOP. Significance statementIn this study, we describe our development of an autologous, closed-loop cell therapy which can migrate to sites of tissue injury and locally secreting an inhibitor of Activin A. Through our use of an Activin A-responsive promoter to drive expression of the recombinant Activin A inhibitor, this engineered cell therapy exhibits closed-loop behavior and effectively prevents heterotopic bone formation in a mouse model of fibrodysplasia ossificans progressiva (FOP). We believe that the findings in this manuscript impactful beyond FOP, and provide a blueprint for the development of marrow-derived cell therapies across the disease spectrum.

Adult zebrafish exhibit scarless repair and functional recovery following spinal cord injury. Their regenerative capacity is attributed to potent stem-like progenitors that mediate neuronal and glial repair. Zebrafish are thought to lack anti-regenerative extracellular matrix (ECM) components abundant in mammalian SCI, but the positive contributions of ECM to spontaneous spinal cord repair are less understood. By employing cross-species single-cell transcriptomics, we found the hyaluran modifying enzyme Hapln1 is upregulated in zebrafish progenitors but not in mouse progenitors following injury. Loss-of-function of hapln1a/b and ablation of hapln1+ cells reduce progenitor cell activation and hinder spontaneous recovery from injury. Using a series of in vivo and in vitro assays, we show that Hapln1 is required for hyaluran-cd44b mediated progenitor cell proliferation. This study reveals that, in addition to lacking anti-regenerative ECM components around SC lesions, zebrafish can also leverage pro-regenerative ECM molecules to enhance progenitor cell potency and promote repair.

In mammals, a dysregulated immune response is detrimental to spinal cord repair. In zebrafish, which are capable of spinal cord regeneration, the immune response promotes regeneration. Neutrophils are the first immune cells to arrive at a spinal cord injury site, but their role in successful regeneration is not fully understood. Here we show that ablating neutrophils, including a subpopulation that expresses the cytokine il4, increases expression of il1b (coding for Il-1{beta}) in macrophages/microglia and impairs anatomical and functional recovery after a spinal cord injury in larval zebrafish. Regeneration is fully rescued by over-expression of il4 alone or experimentally reducing Il-1{beta} levels. Disruption of il4 mimics the detrimental effect of neutrophil ablation for axonal regeneration and is also rescued by reducing Il-1{beta} levels. Hence, after spinal cord injury, a pro-regenerative neutrophil subpopulation promotes spinal cord regeneration in larval zebrafish by controlling expression of il1b in macrophages/microglia. For this neutrophil action, il4 expression is necessary and sufficient. HIGHLIGHTS- Neutrophil ablation impairs spinal cord repair in zebrafish - The neutrophil response can be replaced by reducing Il-1{beta} levels - A pro-regenerative subpopulation of neutrophils expresses il4 - il4 overexpression fully rescues effects of neutrophil ablation

Spinal cord injury (SCI) results in loss of sensory and motor function below the level of damage, with chronic injuries presenting unique challenges for regenerative therapies. While multichannel biomaterial interventions have shown promise in promoting axonal regeneration, circuit restoration, and motor recovery in acute SCI, achieving similar outcomes in chronic injury models remains challenging due to a combination of intrinsic and extrinsic factors. These include the reduced capacity of the neuronal cell body to sustain a growth-activated state and the formation of a physical and chemical barrier at the injury site, preventing axonal growth. To address these challenges and promote motor recovery after chronic injury, we investigated the combinatorial effect of two regenerative approaches: 1) the implantation of poly (lactide-co-glycolide) (PLG) biomaterial bridge to guide axonal growth through the injury site, and 2) the delivery of Epothilone B (EpoB), a microtubule stabilizer that strengthens axons to promote regrowth. We used a transgenic mouse model that selectively expresses a red fluorescent protein variant (tdTomato) reporter throughout the corticospinal tract (CST) under control of the Crym promoter (Crym-tdTomato). We demonstrated that the combination of bridge implantation 60 days after surgical hemisection at C5 with EpoB improved locomotor function. At 12 weeks post-bridge implantation, immunohistology revealed axon regeneration in mice receiving implantation, but not EpoB or no-implant controls. The addition of EpoB significantly increased the volume of both total and CST axons regenerating through the biomaterial channels. Diffusion tensor magnetic resonance imaging (DTI) analysis identified enhanced fractional anisotropy (FA), axial diffusivity (AD), and mean diffusivity (MD) in the bridge region in the combination treatment group, consistent with new intact axons. Furthermore, EpoB enhanced the myelination of regenerated axons in the bridge. Finally, we investigated the proteomic profile of corticospinal neurons ipsilateral and contralateral to the SCI lesion and bridge, comparing the effect of EpoB treatment. Mass spectrometry-based analysis of laser-captured cells in this paradigm identified activation of a regeneration program by corticospinal neurons. These findings present a novel approach to enhance regenerative neural repair and locomotor recovery in chronic SCI.