npj Regenerative Medicine最新文献

The axolotl retains a remarkable capacity for regenerative repair and is one of the few vertebrate species capable of regenerating its brain and spinal cord after injury. To date, studies investigating axolotl spinal cord regeneration have placed particular emphasis on understanding how cells immediately adjacent to the injury site respond to damage to promote regenerative repair. How neurons outside of this immediate injury site respond to an injury remains unknown. Here, we identify a population of dpErk⁺/etv1⁺ glutamatergic neurons in the axolotl telencephalon that are activated in response to injury and are essential for tail regeneration. Furthermore, these neurons project to the hypothalamus where they upregulate the neuropeptide neurotensin in response to injury. Together, these findings identify a unique population of neurons in the axolotl brain whose activation is necessary for successful tail regeneration, and sheds light on how neurons outside of the immediate injury site respond to an injury.

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. CYR61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that has been implicated in fracture repair. Here, we map the distribution of endogenous CYR61 during bone repair and evaluate the effects of recombinant CYR61 delivery on vascularized bone regeneration. In vitro, CYR61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, CYR61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted CYR61-induced neovascularization. Together, these data indicate that CYR61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Despite the therapeutic potential of mesenchymal stromal cells (MSC), there is limited understanding of optimal extracellular matrix (ECM) environments to manufacture these cells. We developed tissue chips to study the effects of multi-factorial ECM environments under manufacturable stiffness ranges and multi-component ECM compositions. Manufacturing qualities of cell expansion potential, immunomodulation, and differentiation capacity were examined. The results show stiffness effects, with 900 kPa substrates supporting higher proliferation and osteogenic differentiation, along with anti-inflammatory IL-10 expression, whereas 150 kPa substrates promoted adipogenic differentiation at 150 kPa, suggesting that optimal ECM environments may differ based on manufacturing goals. ECM biochemistries containing fibronectin and laminin further modulated MSC manufacturing qualities across various stiffnesses. Proteomic and transcriptomic analyses revealed unique ECM combinations that induced higher levels of angiogenic and immunomodulatory cytokines, compared to single factor ECMs. These findings demonstrate that optimized ECM environments enhance MSC manufacturing quality.

Cell replacement therapy is a promising therapeutic option for dry age-related macular degeneration (AMD). In this study, we outline our design for scalable manufacture with appropriate quality gates and present in vivo data for establishing preclinical safety and efficacy of an induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) product, thus laying the foundation for Phase 1/2a trial approval in India (ClinicalTrials.gov ID: NCT06394232; date of registration: 23^rd September 2024). Escalating doses of RPE cell suspension in immunocompromised animals demonstrated absence of tumor formation up to 9 months post-injection. Good Laboratory Practices (GLP) toxicology and tolerability studies in rabbits and non-human primates (NHP) respectively showed no major adverse events. RPE transplanted into immune suppressed RCS rats showed integration, neuroprotection and rescue of visual function. In addition, we provide a detailed description of the modifications in GMP manufacturing protocol to create a final product with a unique composition and Chemistry, Manufacturing and Controls (CMC) studies performed during product development.

Bone fractures and related complications are a significant concern for older adults, particularly with the growing aging population. Therapeutic interventions that promote bone tissue regeneration are attractive for geriatric fracture repair. Extracellular adenosine plays a key role in bone homeostasis and regeneration. Herein, we examined the changes in extracellular adenosine with aging and the potential of local delivery of adenosine to promote fracture healing using aged mice. Extracellular adenosine level was found to be significantly lower in aged bone tissue compared to young mice. Concomitantly, the ecto-5'-nucleotidase CD73 expression was also lower in aged bone. Local delivery of adenosine using injectable, in situ curing microgel delivery units yielded a pro-regenerative environment and promoted fracture healing in aged mice. This study offers new insights into age-related physiological changes in adenosine levels and demonstrates the therapeutic potential of adenosine supplementation to circumvent the compromised healing of geriatric fractures.

Plakophilin-2 (PKP2) mutations cause fatal genetic heart disease and arrhythmogenic cardiomyopathy (ACM) with primary effects on the right ventricle (RV). Adeno-associated virus (AAV)-PKP2 gene therapy shows promise as a therapeutic strategy but lacks long-term data and guidelines on minimal effective doses in animal studies for treating RV deficits, arrhythmia burden, and improving survival when administered during disease settings, which are most relevant to clinical trials. Using AAVrh10, known for its preferential cardiac gene expression at lower doses, we show minimal doses required for efficacy for AAVrh10.PKP2 (LX2020) to rescue cardiac (molecular and especially RV) deficits, arrhythmia burden and survival in PKP2 ACM mice, suggesting its potential to reverse late-stage pathology. Safety assessments in non-human primates revealed no adverse events. These data support LX2020 as a viable treatment for PKP2 ACM patients.

There is an urgent need for innovative therapies targeting defective epithelial repair in chronic diseases like COPD. The mesenchymal niche is a critical regulator in epithelial stem cell activation, suggesting that their secreted factors are possible potent drug targets. Utilizing a proteomics-guided drug discovery strategy, we explored the lung fibroblast secretome to uncover impactful drug targets. Our lung organoid assays identified several regenerative ligands, with osteoglycin (OGN) showing the most profound effects. Transcriptomic analyses revealed that OGN enhances alveolar progenitor differentiation, detoxifies reactive oxygen species, and strengthens fibroblast-epithelial crosstalk. OGN expression was diminished in COPD patients and smoke-exposed mice. An active fragment of OGN (leucine-rich repeat regions 4-7) replicated full-length OGN's regenerative effects, significantly ameliorating elastase-induced lung injury in lung slices and improving lung function in vivo. These findings highlight OGN as a pivotal secreted protein for alveolar epithelial repair, positioning its active fragment as a promising therapeutic for COPD.

Macrophages play a key role in host defense and inflammation, with polarization ranging from pro-inflammatory M1 to anti-inflammatory M2 states. However, effective modulation of macrophage polarity via nucleotide delivery is challenging. This study developed polyethyleneimine-modified carboxyl quantum dots (QDP) as a biocompatible carrier for small RNA delivery to modulate macrophage polarization. QDP-mediated delivery of miR-10a (QDP/miR-10a) rebalanced macrophage polarity and alleviated uterine inflammation and fibrosis in a mouse model of Asherman's syndrome (AS). In vitro, QDP effectively delivered small RNA into RAW 264.7 cells without cytotoxicity, converting LPS-induced M1 to M2 macrophages by inhibiting NF-κB, MAPK, and AKT signaling. In vivo, QDP/miR-10a reduced M1 macrophages, restored polarization, and enhanced uterine restoration in AS mice without affecting systemic immunity. Thus, QDP represents a safe and effective nanocarrier for small RNA delivery to modulate macrophage polarization for inflammatory disease treatment, including AS.

Chronic corneal inflammation, a component of sulfur mustard (SM) and nitrogen mustard (NM) injuries frequently leads to limbal stem cell deficiency (LSCD), which can compromise vision. Corneal conjunctivalization, neovascularization, and persistent inflammation are hallmarks of LSCD. Ocular exposure to SM and NM results in an acute and delayed phase of corneal disruption, culminating in LSCD. Available therapies for mustard keratopathy (e.g., topical corticosteroids) often have adverse side effects, and generally are ineffective in preventing the development of LSCD. We developed a novel, optically transparent HDL nanoparticle (NP) with an organic core (oc) molecular scaffold. This unique oc-HDL NP: (i) markedly improved corneal haze during the acute and delayed phases in vivo; (ii) significantly reduced the inflammatory response; and (iii) blunted conjunctivalization and corneal neovascularization during the delayed phase. These findings strongly suggest that our HDL NP is an ideal treatment for mustard keratopathy and other chronic corneal inflammatory diseases.

We developed acellular tissue engineered vessels (ATEV) using small intestine submucosa (SIS) incorporating heparin and a novel protein named H2R5. ATEVs were implanted into the arterial circulation of an ovine animal model, demonstrating high primary patency rates over a period of three months. Implanted grafts were infiltrated by host cells, the majority of which were monocytes/macrophages (MC/MΦ), as demonstrated by scRNA sequencing and immunostaining. They also developed functional endothelial and medial layers that deposited new extracellular matrix leading to matrix remodeling and acquisition of mechanical properties that were similar to those of native arteries. Notably, during this short implantation time, ATEVs turned into functional neo-arteries, as evidenced by the development of the vascular contractile function. Our findings underscore the potential of H2R5-functionalized ATEVs as promising candidates for tissue replacement grafts in a large pre-clinical animal model and highlight the contribution of macrophages in vascular regeneration.