npj Regenerative Medicine

AO_SCPLOWBSTRACTC_SCPLOWEngineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and the first clinical trial has started in 2021. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage while transplantation in a chronic injury setting was less efficient. We hypothesized that the use of immature EHT patches (EHTIm) could improve cardiomyocytes (CM) engraftment. Chronic myocardial injury was induced in a guinea pig model (n=14). EHTIm (15x106 cells) were transplanted directly after casting. Functional consequences were assessed by serial echocardiography. Animals were sacrificed four weeks after transplantation and hearts were excised for histological analysis. Cryo-injury lead to large transmural scars amounting to 26% of the left ventricle. Grafts were identified by a positive staining for human Ku80 and dystrophin, remuscularizing 9% of the scar area on average. The CM density in the graft was higher compared to previous studies with in vitro-matured EHTs and showed a greater population of immature CM. Echocardiographic analysis showed a small improvement of left ventricular function after EHTIm transplantation. In a small translational proof-of-concept study human scale EHTIm patches (4.5x108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of immature EHT patches without pre-cultivation results in better cell engraftment.

BackgroundRapid closure of open wound, either temporarily or perpetually, is recognized as the standard of care in patients with thermal burns. Human cadaveric allograft and simple genetically modified porcine xenografts are not able to provide enough durable time for extensively burned patients. A selective germline genome edited pig (SGGEP) skin xenograft, Xeno X skin, would be a valuable candidate to the clinical options. MethodsIn an ongoing investigator-initiated clinical trial in patients with thermal burns, the efficacy and safety of cryopreserved Xeno X skin grafts of SGGEP for burned patients were evaluated. Each patient received surgical grafting with a skin xenotransplant and wild type pig extracellular matrix (wpECM) in a side-by-side manner for in-situ comparison. The primary outcome measures of xeno-skin grafts included Xeno X skin safety and tolerability, as well as the quality and duration of temporary barrier function yielded by Xeno X skin grafts (as determined by Baux score). Seven parameters included in the analysis were vascularization, pigmentation, thickness, relief, pliability, surface area and the overall opinion, with each calculated on an independent 0-10 scale. ResultsA total of 16 burned patients completed the trial. All the patients tolerated Xeno X skin grafts well and no advent events were observed. In all cases, Xeno X skin grafts were vascularized and fully adherent, they also exhibited better overall outcomes than those of wpECM. Xeno X skin grafts survived for at least 25 days without a need of any immunosuppressive drug, well consistent with our earlier preclinical studies in non-human primates. ConclusionXeno X skin grafts of SGGEP did not incur any signs of local and systemic safety issues, and in the meanwhile provided a high quality and long duration of temporary barrier function for burned patients. This is a major milestone in the xenotransplant field, indicating that genome-edited organ xenotransplant has become a clinical reality.

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.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, we evaluated the differences between the responses of MRL/MpJ versus C57BL/6 strain in traumatic muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury was a robust adipogenesis within the muscle. This was associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there were fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model could provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and materials implantation.

End-stage lung disease and lung cancer significantly contribute to global mortality, necessitating new research strategies for studying pulmonary biology. Here, we present an engineered whole-lung tissue model used to evaluate the effects of cellular communities on tissue organization and alveolar barrier function. Engineered lungs were grown ex vivo on decellularized whole-lung matrices as structurally biomimetic, bioactive scaffolds. Histologic architecture of engineered lungs improved with the addition of alveolar macrophages, coming to resemble neonatal lung. Incorporating alveolar macrophages maximized the differentiation of native-like cellular communities, including alveolar type I-like epithelium, bronchioalveolar stem cells, microvascular endothelium, and pericytes. Cell-cell signaling in engineered lungs showed activation of developmental and inflammatory pathways, including WNT, Notch, and FGF signaling pathways. Engineered lungs containing alveolar macrophages showed a 668% improvement in measured alveolar barrier function. This work demonstrates the potential utility of engineered lung models for studying principles of tissue biology and pulmonary regeneration.

During wounding and material implantation there is a disturbance in tissue homeostasis and release of self-antigen, and regulation between tolerance and auto-inflammation in injury is not well understood. Here, we analyzed antigen-presenting cells in biomaterial-treated muscle injury and found that pro-regenerative materials enrich Batf3-dependent CD103+XCR1+CD301b+ dendritic cells associated with cross-presentation and self-tolerance. Muscle trauma was accompanied by CD8+ iTregs and expansion of CD103+XCR1+CD62L- adaptive immune cells. Up-regulation of E-Cadherin (the ligand for CD103) and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Without cross-presenting cells T cell activation increases, pro-regenerative macrophage polarization decreases, and muscle healing is impaired. These data describe a regulatory communication network through CD103+XCR1+ immune cells resulting in downstream effects on tissue regeneration.

Heart regeneration requires coordinated immune activation, timely inflammatory resolution, and dynamic extracellular matrix (ECM) remodeling in addition to cardiomyocyte (CM) proliferation. However, the cytokine signals that instruct immune cell functions during cardiac repair remain incompletely understood. Here, we identify interferon-gamma (IFN-{gamma}) as a critical regulator of macrophage plasticity in zebrafish heart regeneration. IFN-{gamma} signaling components are dynamically activated following cardiac injury, with early induction of ifng1 and temporally coordinated receptor expression. Genetic ablation of ifng1 impairs myocardial regeneration, resulting in reduced CM proliferation and persistent fibrotic scarring. Temporal transcriptional profiling reveals sustained inflammatory signatures, impaired efferocytosis, and abolished reparative programs, accompanied by aberrant immune cell dynamics and retention of injury-derived debris in mutant hearts. Transcriptomic analysis of cardiac macrophages further reveals that IFN-{gamma} deficiency disrupts the transition from an inflammatory state to a reparative, ECM-remodeling phenotype, leading to reduced collagen denaturation and diminished CM protrusion at the injury border zone. Inducible- and macrophage-specific blockade of IFN-{gamma} signaling phenocopies defects in global knockout, establishing a cell-autonomous requirement for IFN-{gamma} in coordinating regenerative immune function. Collectively, our findings define an IFN-{gamma}-dependent macrophage reprogramming axis that couples inflammatory resolution to ECM remodeling in heart regeneration, elucidating how cytokine signaling actively instructs tissue repair. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/712551v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@cefbecorg.highwire.dtl.DTLVardef@fd56dborg.highwire.dtl.DTLVardef@517495org.highwire.dtl.DTLVardef@1bd0851_HPS_FORMAT_FIGEXP M_FIG C_FIG

Flexor tendon injuries are common and heal poorly owing to both the deposition of function-limiting peritendinous scar tissue and insufficient healing of the tendon itself. Therapeutic options are limited due to a lack of understanding of the cell populations that contribute to these processes. Here, we identified the epitenon as a major source of cells that contribute to both peritendinous fibrosis and regenerative tendon healing following acute tendon injury. Using a combination of genetic lineage tracing and single cell RNA-sequencing (scRNA-seq), we profiled the behavior and contributions of each cell fate to the healing process in a spatio-temporal manner. Integrated scRNA-seq analysis of mouse healing with human peritendinous scar tissue revealed remarkable transcriptional similarity between mouse epitenon-derived cells and fibroblasts present in human peritendinous scar tissue, which was further validated by immunofluorescent staining for conserved markers. Finally, ablation of pro-fibrotic epitenon-derived cells post-tendon injury significantly improved functional recovery. Combined, these results clearly identify the epitenon as the cellular origin of an important progenitor cell population that could be leveraged to improve tendon healing.

A balanced immune response after cardiac injury is crucial to successful heart regeneration, but knowledge of what distinguishes a regenerative from a scarring response is still limited. The Mexican cavefish provides a unique comparative model to study heart regeneration and scarring within a single species. Surface-dwelling fish are capable of heart regeneration whereas their cave-dwelling Pachon counterparts lack this ability, similar to the human heart. Using single-cell transcriptomics and immune perturbations, we find significant differences in the immune response between the two populations. Unlike the transient response in the scarring Pachon, the regenerative surface fish heart generates an unexpected functionally active prolonged innate and adaptive immune response at the late stages of regeneration. Inhibiting the overall prolonged immune response impairs regeneration and cardiomyocyte proliferation. Further characterisation of specific cell types shows that late-present macrophages are phagocytic, and their depletion disrupts regeneration but not cardiomyocyte proliferation while inhibiting B cells impairs regeneration by reducing cardiomyocyte proliferation. This B cell response is conserved in zebrafish. Our findings reveal critical immune mechanisms distinguishing regenerative and non-regenerative responses, offering insights for potential therapeutic strategies to enhance heart repair.

Heart left or right ventricular failure results from either ischemic or congenital diseases, respectively, and remains a major health burden in our societies. There is thus a high demand for a regenerative therapy. Yet, the ability of the adult post-mitotic mammalian heart to self-regenerate remains largely a challenge Here, we combined cell therapy in a pig with right heart failure, cardiac physiology, single cell RNA-seq and spatial transcriptomics. We demonstrate that resident cardiac macrophages mediate a process of cardiomyocytes de-differentiation to form a blastema which produces new proliferative cardiomyocytes. Thus, a mammalian heart close to a human heart features the ability to undergo epimorphosis and to regenerate. A direct and specific target of resident macrophages holds promise to regenerate hearts, specifically in a growing population of now adult congenital heart diseases patients with right ventricular failure and left without any efficient pharmacological relieving treatment.

Balancing between regenerative processes and fibrosis is crucial for heart repair, yet strategies regulating this balance remain a barrier to developing therapies. While Interleukin11 (IL11) is known as a fibrotic factor, its contribution to heart regeneration is poorly understood. We uncovered that il11a, an Il11 homolog in zebrafish, can trigger robust regenerative programs in zebrafish hearts, including cardiomyocytes proliferation and coronary expansion, even in the absence of injury. However, prolonged il11a induction in uninjured hearts causes persistent fibroblast emergence, resulting in fibrosis. While deciphering the regenerative and fibrotic effects of il11a, we found that il11-dependent fibrosis, but not regeneration, is mediated through ERK activity, suggesting to potentially uncouple il11a dual effects on regeneration and fibrosis. To harness the il11as regenerative ability, we devised a combinatorial treatment through il11a induction with ERK inhibition. This approach enhances cardiomyocyte proliferation with mitigated fibrosis, achieving a balance between regenerative processes and fibrosis. Thus, we unveil the mechanistic insights into regenerative il11 roles, offering therapeutic avenues to foster cardiac repair without exacerbating fibrosis.

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.

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.

In the mouse distal terminal phalanx (P3), it remains unclear why amputation at less than 33% of the digit results in regeneration, while amputation exceeding 67% leads to non-regeneration. Unraveling the molecular mechanisms underlying this disparity could provide crucial insights for regenerative medicine. In this study, we aim to investigate the tissues within the wound bed to understand the tissue microenvironment associated with regenerative versus non-regenerative outcomes. We employed a P3-specific amputation model in mice, integrated with time-series RNA-seq and a macrophage assay challenged with pro- and anti-inflammatory cytokines, to explore these mechanisms. Our findings revealed that non-regenerative digits exhibit a greater intense early transcriptional response in the wound bed compared to regenerative ones. Furthermore, early macrophage phenotypes differ distinctly between regenerative and non-regenerative outcomes. Regenerative digits also display unique co-expression modules related to Bone Morphogenetic Protein 2 (BMP2). The differentially expressed genes (DEGs) between regenerative and non-regenerative digits are enriched in targets of several transcription factors, such as HOXA11 and HOXD11 from the HOX gene family, showing a time-dependent pattern of enrichment. These transcription factors, known for their roles in bone regeneration, skeletal patterning, osteoblast activity, fracture healing, angiogenesis, and key signaling pathways, may act as master regulators of the regenerative gene signatures. Additionally, we developed a deep learning AI model capable of predicting post-amputation time and level from RNA-seq data, with potential applications in personalized treatment strategies and assessing the impact of interventions on regenerative outcomes.

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

Vascular graft fibrosis can cause a decrease in cellular infiltration and capillary ingrowth in vascular walls and vascular stiffening. As such, there are still no vascular grafts that can be used in blood vessels where their diameters are less than 6 mm in patients. Although various approaches have been evaluated to mitigate implant-associated fibrosis, effective treatments remain quite limited. In this study, we demonstrated that APOE was significantly increased during vascular regeneration after graft implantation in vivo. APOE knockout (KO) increased compliance of regenerated aortas and reduced extracellular matrix (ECM) deposition in adventitia of the regenerated aortas. Using single cell RNA sequencing (scRNA-seq), a subset of profibrotic macrophages was found to be involved in graft fibrosis and APOE KO limited the formation of profibrotic macrophage formation during vascular regeneration. The interaction between APOE and low-density lipoprotein receptor related protein 1 (LRP1) partially mediated fibrotic differentiation of the macrophages. Profibrotic macrophages promoted graft fibrosis mainly through secretion of insulin-like growth factor-1 (IGF-1) that could support proliferation of fibroblasts. Finally, we showed that APOE knockdown in vivo using adeno-associated virus (AAV) improved the compliance of regenerated aortas and reduced ECM deposited in the adventitial areas by limiting formation of profibrotic macrophages. Collectively, these data indicate that APOE promotes the profibrotic transition of macrophages partially through LRP1, and the profibrotic macrophages increase the proliferation of fibroblasts via IGF-1. Inhibition of APOE by AAV can alleviate graft fibrosis occurring during vascular regeneration.

Muscle regeneration is associated with transient induction of cellular senescence. However, the role of senescence in muscle regeneration of young mice remains unclear. Using a mouse model deficient in both Cdkn1a and Cdkn2a, we find that a marked reduction in senescent cells correlates with delayed muscle regeneration. Single-cell RNA sequencing reveals a heterogeneous senescence program composing of multiple cell types. Notably, senescent fibro-adipogenic progenitors (FAPs) upregulate Mcl-1 to acquire apoptosis resistance. Moreover, removing senescent FAPs using a Mcl-1 inhibitor S63845 impairs muscle regeneration. Furthermore, we find that senescent FAPs promotes myogenic differentiation in a paracrine manner. Hence, these results highlight the beneficial role of senescent stromal cells in supporting muscle regeneration.

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.

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

Summary ParagraphMammalian skin appendages, such as hair follicles and sweat glands, are complex mini-organs formed during skin development1, 2. As wounds heal, the resulting scar tissue lacks skin appendages. The clinical regeneration of skin appendages is an ongoing challenge3, 4. Skin epithelial tissues have been regenerated in vivo by cellular reprogramming5, 6, but the de novo generation of skin appendages has not previously been achieved. Here, we show that transplantation of a type of epithelial cell and two types of mesenchymal cells, reprogrammed from adult mouse subcutaneous mesenchymal cells to mimic developing skin cells, resulted in the generation of skin-appendage-like structures. Furthermore, with the development of a new AAV serotype, in vivo reprogramming of wound-resident cells with the same reprogramming factors generates skin with de novo appendages in adult mice. These findings may provide new therapeutic avenues for skin regeneration and frequent aging-associated skin appendage disorders, such as hair loss and dry skin, and may extend to other tissues and organs. This study also provides the potential for de novo generation of complex organs in vivo.