npj Regenerative Medicine最新文献

Annually, 10% of 15 million bone fractures in the US fail to heal, and fractures with compromised blood flow, i.e., ischemia, are five times more likely to become nonunions. While ischemia is known to impair healing, the cellular and molecular mechanisms underlying this deficiency are unclear. Wild-type mice with surgically-induced ischemia underwent tibia fractures, and single-cell RNA-sequencing was performed on calluses at days 4 and 7 post-fracture. We observed delayed chondrogenic differentiation and upregulation of Cyclin-Dependent Kinase 8 (Cdk8) by stromal progenitors and fibroblasts in the ischemic callus. Hypoxia induced CDK8 gene expression in human mesenchymal stromal cells (hMSC), and pharmacological CDK8 inhibition promoted hMSC chondrogenic and osteogenic potential. In vivo oral delivery of a CDK8 inhibitor enhanced callus chondrogenesis and mineralization, potentially improving ischemic fracture healing. Our results suggest that CDK8 impedes stromal cell differentiation and that its inhibition may be a clinically translatable approach to enhance ischemic fracture healing.

Neurogenic bladder (NB) is a disabling condition lacking effective therapies. This study investigated whether CD73-expressing adipose-derived stem cells (ADSCs) promote bladder repair in a rat model of NB and explored the underlying mechanisms. ADSCs were sorted into CD73⁺ and CD73⁻ populations, and CD73⁺ cells were further modified to generate CD73⁺^/ev ADSCs and CD73-overexpressing CD73⁺^/⁺ADSCs, while CD73 inhibition was achieved using APCP. Conditioned media were applied to rat bladder smooth muscle cells in vitro, and ADSCs were injected into the bladder wall of rats subjected to bilateral pelvic nerve crush. Four weeks after treatment, bladder function, histology, and molecular markers were evaluated. CD73 overexpression enhanced VEGF and SDF-1 expression, promoted cell proliferation, and reduced inflammatory cytokines, whereas APCP suppressed VEGF. In vivo, CD73⁺^/⁺ADSCs improved cystometric parameters, regenerated bladder tissue, reduced pyroptosis, and activated the PI3K/AKT/mTOR pathway, while suppressing NF-κB/NLRP3/caspase-1 signaling. CD73 expression and VEGF progressively declined in untreated NB rats but were restored by CD73⁺^/⁺ADSCs. These findings indicate that CD73 enhances ADSC-mediated bladder repair through dual pro-regenerative and anti-inflammatory actions, suggesting a promising therapeutic strategy for NB.

The repair of corneal injuries remains a major challenge in clinical practice. Impaired corneal wound healing is closely associated with aberrantly activated stromal keratocytes and disorganized extracellular matrix. Here, we identify aberrant Hedgehog signaling in corneal keratocytes as a key driver of defective wound repair. In adult mice, Hedgehog signaling is suppressed in quiescent keratocytes but is pathologically reactivated following chemical injury, correlating with impaired repair. Keratocyte-specific Hedgehog activation via Ptch1 ablation disrupted corneal wound healing after epithelial scraping-a process that would normally resolve seamlessly under physiological conditions. Mechanistically, Hedgehog activation induced stromal thinning and stiffening through disorganized collagen fibrils. Transcriptomics analysis revealed keratocyte transdifferentiation into fibroblast-like phenotypes, accompanied by downregulation of extracellular matrix genes. Hedgehog-mediated stromal stiffening suppressed YAP activity in the overlying epithelium via Hippo pathway activation, blocking epithelial differentiation-a defect that was reversed by Hippo inhibition (XMU-MP-1). In chemical injury models, genetic Smo deletion or pharmacological Gli1/2 inhibition (GANT61) restored stromal architecture, normalized collagen organization, and rescued epithelial differentiation defects. These findings establish Hedgehog signaling in keratocytes as a critical regulator of stromal-epithelial crosstalk and highlight its targeted inhibition as a potential therapeutic strategy to restore corneal transparency and repair fidelity after injury.

Children with congenital heart defects increasingly survive to adulthood, but the non-physiological Fontan circulation imposed by current surgical palliation leads to significant sequelae and reduced lifespan. Restoring subpulmonic pumping function remains a long-standing goal, and there have been several attempts using regenerative medicine approaches. These efforts have lacked biomechanical rigor, however, and have not achieved the requisite functionality. Here, we introduce an analytically based framework that grounds pulsatile conduit design in biomechanical principles, coupling the architecture and properties of a passive matrix with embedded myofibers to optimize performance within pediatric anatomical constraints. Parametric exploration of matrix properties and myofiber orientations yields biomechanically feasible designs. Sensitivity analyses demonstrate design robustness and highlight parameters critical for reproducible biomanufacturing and surgical implementation. To illustrate clinical potential, a patient-specific lumped-parameter hemodynamic model shows that an optimized pulsatile conduit can generate physiologically meaningful pressures and flows and outperform passive grafts.

Muscle function and regeneration are impaired in type 1 diabetes, but whether this arises directly from muscle stem cell (MuSC) dysfunction has not been addressed. Here, we utilized three-dimensional MuSC cultures (micromuscles) to demonstrate that hyperglycemia drives deficits in muscle stem cell function, leading to impaired force production in differentiated myotubes. The functional capacity of skeletal muscle was shown to decline after repeated bouts of injury in mouse models of type 1 diabetes, and this was replicated in micromuscles derived from MuSCs isolated from diabetic mice, indicating MuSC dysfunction was linked to poor muscle regeneration and function. The loss of force producing capacity was associated with impaired myotube hypertrophy in vitro and in vivo after injury. Furthermore, poor muscle regeneration was exacerbated by a loss of MuSC number due to aberrant activation, even in the absence of injury. Deficits in MuSC function and number could be rescued by early treatment with the glucose-lowering drug dapagliflozin, indicating that MuSC defects were driven by exposure to a hyperglycemic environment. The findings reveal that MuSC dysfunction contributes to muscle functional deficits in models of type 1 diabetes.

Trans-sutural distraction osteogenesis (TSDO) is an effective treatment of midfacial hypoplasia, a craniofacial deformity frequently associated with cleft lip and palate. Though extracellular matrix (ECM) remodeling plays a pivotal role in craniofacial correction, the characteristics and mechanisms underlying collagen reorganization and cellular morphological adaptations during TSDO remain poorly understood. This study quantitatively delineates the spatiotemporal changes of sutural cells and ECM morphology, revealing a polarized alignment parallel to the direction of mechanical force. Multi-omics analysis demonstrates that macrophages regulate collagen remodeling in suture mesenchymal stem cells (SuSCs) via the PDGF signaling pathway. Subsequent in vitro stretch loading models confirmed PDGF pathway activation enhances SuSCs migration, collagen synthesis, and cellular morphological reorganization. Validation in macrophage-elimination murine models further corroborated this regulatory axis. Collectively, our work maps the dynamic microenvironmental changes during TSDO and elucidates cell-cell interaction-driven ECM collagen remodeling. These insights advance the understanding of TSDO-mediated osteogenesis and provide a foundation for developing optimized therapeutic strategies.

Leading gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin (µDys) have shown partial efficacy in patients, contrasting with the favorable outcomes observed in animal models. The identification of effective therapeutic strategies could be accelerated by using human high-throughput DMD models that replicate the molecular complexity driving pathology for accurate screening. To face this challenge, we develop MYOrganoids, an engineered muscle platform derived from patient-induced pluripotent stem cells (iPSC), recapitulating critical hallmarks of DMD, such as fibrosis and muscle dysfunction. We show that co-culture of fibroblasts with iPSC-derived muscle cells during organoid generation is pivotal for functional maturation and muscle force evaluation upon eccentric contractions. Notably, incorporation of DMD fibroblasts induced phenotypic exacerbation in DMD MYOrganoids by unraveling of fibrotic signature and fatiguability through cell-contact and paracrine mechanisms. We then exploited our system to interrogate gene therapy efficacy in this severe context. Although µDys gene transfer improves muscle resistance and partially restores membrane stability, it fails to reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our system, a limitedly explored aspect in DMD models, and provide the opportunity to study mechanisms of dysregulated cellular communication and empower gene therapy efficacy.

Pelvic organ prolapse (POP) due to weak support tissues is a common, debilitating condition typically treated with surgery. However, surgery is suboptimal due to associated risks and high prolapse recurrence rates. Therefore, there is a need for non-surgical therapies to restore supportive tissues, such as the vagina, following surgical intervention. In this study, we used patient induced pluripotent stem cells as a source to generate patient-specific progenitors of smooth muscle cells (pSMCs) and collected secretomes from these progenitor cells to examine their paracrine effects. Proteomic analysis of the conditioned media from pSMCs (pSMC-CM), which contain the secretomes, revealed proteins involved in extracellular matrix (ECM) remodeling. We assessed the paracrine effect of pSMC-CM using vaginal fibroblasts from POP patients and in a rat model of surgically injured vagina. pSMC-CM increased ECM protein expression in human vaginal fibroblasts and enhanced vaginal contractile function and ECM protein deposition in the surgically injured rat vagina. These findings suggest that pSMC-CM may promote vaginal contractile function and tissue extracellular matrix remodeling following surgical intervention.

Vascular network disruption caused by spinal cord injury (SCI) exacerbates secondary neuronal damage. Although vascular barrier disruption naturally restores over time, its underlying mechanism is not fully clarified. Here, we found that blood factors promote the proliferation of endothelial cells (ECs), which are essential for revascularization in the repair process after SCI. In vivo, endogenous IgG leakage into the spinal cord initiates EC proliferation at the lesion after injury. In vitro, adult mouse serum promotes mouse ECs proliferation through beta-2-microglobulin (B2M) via transforming growth factor beta receptor 2 (TGFBR2). Under EC-specific knockdown of Tgfbr2 in vivo, we observed exacerbated blood extravasation and increased inflammatory cell infiltration compared to controls. Additionally, suppression of endothelial Tgfbr2 impaired motor function recovery, axon regrowth, and regeneration in injured mice. These findings suggest that targeting the B2M-TGFBR2 axis could be a potential therapeutic approach to promoting functional recovery against vascular disruption after SCI.

Induced pluripotent stem cells (iPSCs) have demonstrated superior capacity to regenerate hyaline cartilage compared to mesenchymal stromal cells (MSCs). However, most previous animal studies have only conducted short-term assessments. We performed a long-term (8 weeks) in vitro chondrogenesis of human iPSC-derived multipotent progenitor cells (iMPCs) and human MSCs. The expression levels of hypertrophy-related genes were significantly lower in the iMPC group compared to the MSC group, such as collagen type X being 5-fold lower on day 56. In the animal study, implants from the iMPC group maintained more matrix than the MSC group at both short and long-term time points (12 and 48 weeks). Importantly, at 48 weeks, the native cartilage surrounding the defect areas in some rats from the MSC group showed severe degradation, which was not observed in the iMPC group. In conclusion, iMPCs represent a safe and effective cell source for long-term hyaline cartilage repair.