Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host–graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.

Background: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). Methods: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. Results: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. Conclusion: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM‐MSC), human immortalized MSC (hTERT‐MSC), human embryonic stem cells (ESC), ESC‐derived cardiac progenitor cells (CPC), human ESC‐derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC‐derived sEV (ESC‐sEV) exhibited the best pro‐angiogenic and anti‐fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI‐reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC‐sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down‐regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT‐MSC, ESC, and CPC revealed factors in ESC‐sEV that potentially drove the observed functions. In conclusion, ESC‐sEV holds great promise as a cell‐free treatment for promoting cardiac repair following MI.

Formyl peptide receptor 2 (FPR2) is a G protein–coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca²⁺ and release NADPH oxidase–derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit β-arrestin and induce neutrophil chemotaxis, supporting the previous notion that β-arrestin translocation is of importance for cell migration. Despite the lack of β-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit β-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca²⁺, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of β-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections. Key Points FPR2-specific PSMα peptides are not chemotactic but activate the NADPH oxidase. PSMα peptides induce ERK1/2 phosphorylation but not β-arrestin recruitment.

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

Targeted delivery of therapeutic agents is a persistent challenge in modern medicine. Recent efforts in this area have highlighted the utility of extracellular vesicles (EVs) as drug carriers, given that they naturally occur in bloodstream and tissues, and can be loaded with a wide range of therapeutic molecules. However, biodistribution and tissue tropism of EVs remain difficult to study systematically. Here, a multiplexed approach is developed for simultaneous tracking of EVs from various cell lines within a single in vivo experiment. EVs are used from 16 different cell lines, and through controlled fusion with lipid nanoparticles (LNPs) carrying single‐stranded DNA barcodes, uniquely barcoded hybrid EV particle (hEV) library is generated. These hEVs are combined for a multiplexed in vivo biodistribution profiling in mice, and discovered that HAP1‐derived hEVs demonstrated lung tropism, suggesting that these hEVs may be used for targeted drug delivery into lung tissue. To examine this possibility further, it is shown that HAP1 hEV loaded with Cre mRNA displayed functional delivery to the lungs. Overall, the barcoded hEV technology enables rapid profiling of biodistribution across EV cell sources, which is poised to improve throughput and extent of EV studies, while reducing the number of animals required for research.