Transition of extracellular vesicle (EV) research from basic discovery to clinical application raised hopes regarding diagnostic, therapeutic and prognostic progress. Rigorous reporting of experimental details is required to align the EV field with pharmaceutical quality standards. MISEV2023 recommendations encourage concise reporting but widespread adoption remains limited. Current reporting tools are time-consuming, and adherence declines despite rapidly growing number of EV studies. We therefore created EV-Checklist, a complementary digital tool that streamlines reporting and increases transparency. By uploading manuscript text, an AI-assisted algorithm automatically completes a checklist covering EV nomenclature, source, isolation, characterization and function. To ensure accuracy, users validate AI-generated entries before submission-ideally, gaps in reporting can be closed (e.g., missing particle/protein ratio). The resulting concise report can accompany manuscripts helping editors, reviewers and readers by presenting key methodological and results details at a glance. EV-Checklist complements existing comprehensive registries as 'fast-and-easy' tool enhancing clarity and accessibility of EV research data and may promote higher adherence to documentation standards. Adoption may be encouraged by journal endorsement to streamline the review process for compliant submissions, signalling adherence to MISEV2023 standards. EV-Checklist and an accompanying AI-assisted search tool (PMC EV Search), spanning over 45,700 open-access EV manuscripts, are publicly available at https://ev-zone.org/.

Introduction:Successful bone regeneration after dental and craniofacial reconstruction can present a significant clinical challenge. In this study, we investigated whether the regenerative potential of human periosteum-derived stromal progenitor cells (PDCs) depends on their anatomical origin. Methods:Paired periosteum samples were collected from jaw and appendicular bones (fibula, femur, or iliac crest) of five donors and expanded in a xeno-free, platelet lysate-supplemented medium. All cultures exhibited a typical fibroblastic morphology and a uniform stromal immunophenotype (≥95% CD73/90/105 and ≤2% CD45/14/19/HLA-DR). PDCs were tested in a 3D cartilage disc formation assay after seeding on collagen-coated transwell inserts or on transferrable porcine collagen membranes. Results:Appendicular-derived PDCs consistently formed stable, cartilage-like 3D discs with a mean weight of 14.7 mg and a range of 8.8-23.6 mg, whereas jaw-derived PDCs produced contracted, irregular aggregates. Histological and molecular analyses confirmed greater proteoglycan deposition and higher COL2A1 expression in appendicular-derived PDCs, indicating superior intrinsic chondrogenic potential. Culturing jaw-derived PDCs on porcine collagen membranes did not enhance their differentiation capacity. Conclusion:Our findings demonstrate that the chondrogenic potential of PDCs is strongly determined by their anatomical origin. Therefore, selecting the appropriate periosteal source, combined with optimized biomaterials, is crucial for achieving predictable and efficient bone regeneration.

Gene therapies offer new possibilities for the precise correction of monogenic disorders. Here, we present the first prime editing (PE)-based gene repair strategy for pathogenic COL17A1 variants that cause junctional epidermolysis bullosa (JEB). Type XVII collagen (C17), encoded by COL17A1, plays a critical role in skin aging, regeneration, and the maintenance of epidermal stem cell integrity. Treatment of primary human JEB keratinocytes with PE mRNAs resulted in COL17A1 editing efficiencies of up to 60% in bulk-treated cells, leading to the restoration of full-length, accurately shed C17. Chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing analysis of gene-edited JEB keratinocytes confirmed the absence of unintended chromosomal rearrangements at potential off-target sites and only minimal on-target aberrations. Remarkably, in a xenograft model, in which C17+ cells represented only 55.9% of the input population, COL17A1-corrected cells populated 92.2% of the basal keratinocyte layer in the resulting skin grafts after 6 weeks. These observations highlight a potential selective advantage imparted by C17 restoration, in line with its canonical role in anchoring hemidesmosomes to the basement membrane and preserving the structural integrity of the interfollicular epidermal stem cell niche. Based on our results, we envision PE as an efficient and safe option to restore gene function in EB and other genodermatoses.

BACKGROUND:The converging biology between enveloped viruses and extracellular vesicles (EVs) has raised interest in the application of engineered EVs as antiviral therapeutics. Following the recent COVID-19 pandemic, EVs engineered with either the ACE2-receptor or Spike-protein have been proposed as strategy to either decoy SARS-CoV-2, or to compete with its cell entry. For generic use as a platform for future pandemic preparedness, a systematic and quantitative comparison of both strategies is required to assess their limitations and benefits across different variants of concern. METHODS:Here we generated EVs decorated with either the ACE2-receptor or the Spike-protein of (Wuhan)-SARS-CoV-2 and used single vesicle imaging for in-depth quantitative characterisation. These vesicles were then systematically tested for anti-viral activity across SARS-CoV-2 variants of concern using both, pseudotype and live virus cellular infection models including primary human bronchial and nasal explants. RESULTS:Spike-protein EVs or ACE2-EVs recovered from transiently transfected HEK293T cells comprised only a small fraction of the EV secretome (5% or 20%, respectively) and were primarily derived from the plasma membrane rather than multivesicular bodies. Redirecting intracellular trafficking of the Spike protein by mutating its transmembrane or subcellular localisation domains did not increase the yields of Spike-EVs. Both types of vesicles inhibited SARS-CoV-2 (D614G) in a dose dependent manner with kinetics and immunohistochemistry consistent with an inhibition at the initial cell entry stage. ACE2-EVs were more potent than Spike-EVs and at least 500-1000 times more potent than soluble antibodies in a pseudotype model. Surprisingly, ACE2-EVs switched from an inhibitory to an enhancer activity for the Omicron BA.1 variant whereas Spike-EVs retained their activity across all variants of concern. CONCLUSIONS:While our data show that both types of engineered EVs potently inhibit SARS-CoV, the decoy versus competition strategy may result in diverging outcomes when considering viral evolution into new variants of concern. While Spike-EVs retain their competition for receptor binding even against higher affinity viral Spike mutations, the formation of complexes between ACE2-EVs and the virus may not only result in inhibition by decoy. As EVs are actively internalised by cells themselves, they may shuttle the virus into cells, resulting in a productive alternative cell entry route for variants such as Omicron, that diverge from strict plasma membrane protease cleavage to the use of endosomal proteases for release of their genome.

Acute myeloid leukaemia (AML) is a haematologic malignancy with high relapse incidence and mortality. Approximately one-third of AML patients carry an fms-like tyrosine kinase 3 (FLT3) mutation, often associated with GLI expression and Hedgehog signalling. AML cells shape their microenvironment into a leukaemia-permissive space by releasing extracellular vesicles (EVs). EVs can transfer chemoresistance and thereby play an important role in refractory and relapsing diseases. Here, we discovered a synergistic effect of combined treatment with the FLT3 inhibitor Crenolanib and the Hedgehog pathway inhibitor HPI-1 in the AML cell lines MOLM-14 and MV4-11. In-depth comparative proteomics revealed alterations in the cellular and the EV proteome upon single or combined inhibition of FLT3 and GLI, highlighting affected pathways. By comparing cellular and EV proteomes, we found that transport of ribosomal proteins, such as RPS26 and RPL27A, and ErbB pathway members such as GAB1, GRB2 and SHC1 to EVs, is selectively avoided upon treatment with Crenolanib. These findings were corroborated by comparative proteomics of EVs derived from AML patients and healthy donors. Ribosomal and ErbB signalling pathway proteins may play an important role in microenvironmental modulation by EVs, and Crenolanib treatment potentially acts by interfering with leukaemia niche formation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) constitute a promising therapy for myocardial infarction (MI). The lack of an effective immunosuppressive regimen, combined with single-cell transplantations, results in suboptimal outcomes, such as poor engraftment and compromised therapeutic efficacy. This study aimed to confirm the increased retention of hiPSC-CMs microtissues (CMTs) over single-cell grafts. To ensure the long-term survival of CMTs for potential cardiac applications, CMTs were transplanted in a porcine model of MI using a triple immunosuppression protocol designed to limit immune cell infiltration. Acute evaluation of spherical hiPSC-CMs aggregates and dissociated aggregates followed by the development of a triple immunosuppression protocol were performed in healthy animals. Long-term survival of CMTs was later examined in pigs that underwent a transient coronary occlusion. Two weeks post-MI, the immunosuppression treatment was initiated and on day 28 the animals were transplanted with CMTs and followed for four more weeks. Acutely, CMTs showed superior retention compared to their dissociated counterparts. The immunosuppression regimen led to no organ damage and stable levels of circulating drugs once optimal dose was achieved. Two weeks post-xenotransplantation in healthy pigs, histology revealed that immunosuppressed animals displayed a significant decrease in total cellular infiltrates, particularly in CD3 + T cells. Pigs that underwent coronary occlusion, which later were immunosuppressed and treated with CMTs (5 × 10 7 cells), showed cell engraftment onto the native myocardium four weeks post-transplantation. This study supports the use of a triple immunosuppression cocktail to ensure long-term survival of CMTs for the treatment of MI. Graphical Abstract In vivo evaluation of CMT transplantation as a regenerative therapy for myocardial infarction. Cardiac microtissues are potential therapies that, when administered in immunosuppressed pigs, have the potential to survive long-term and remuscularize the infarcted myocardium. Figure created with https://BioRender.com .

BACKGROUND:While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target.OBJECTIVES:We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC.METHODS:We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo.RESULTS:Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival.CONCLUSIONS:Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Extracellular vesicle (EV) research has experienced rapid growth in the past decade. While community-driven efforts, such as the MISEV guidelines (2014, 2018, 2023), have aimed to standardize reporting and enhance experimental rigor, these have not yet been universally adopted in daily EV research practices. The complexity and time commitment required by existing reporting tools can deter researchers from fully embracing them, leading to incomplete or inconsistent documentation in EV studies. Therefore, we created EV-checklist, a complementary digital tool to streamline documentation during manuscript submission and increase transparency for the reviewers, editors and readers. Our tool guides researchers through a straightforward checklist covering nomenclature, source, isolation, characterization, and functional studies of EVs. It generates a concise two-page PDF formatted table for easy integration into manuscripts and provides editors, reviewers, and readers with a clear overview of the conducted studies. EV-Checklist intends to complement existing comprehensive registries as a ‘fast-and-easy’ tool to enhance clarity and accessibility of methodological and functional insights. It may also promote higher adherence to documentation standards and in EV studies. Our tool is available on . ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Acute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood. METHODS:Public genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML. RESULTS:Elevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms. CONCLUSION:Our findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients.

The evolution of extracellular vesicle (EV) research has introduced nanotechnology into biomedical cell communication science while recognizing what is formerly considered cell "dust" as constituting an entirely new universe of cell signaling particles. To display the global EV research landscape, a systematic review of 20 364 original research articles selected from all 40 684 EV-related records identified in PubMed 2013-2022 is performed. Machine-learning is used to categorize the high-dimensional data and further dissected significant associations between EV source, isolation method, cargo, and function. Unexpected correlations between these four categories indicate prevalent experimental strategies based on cargo connectivity with function of interest being associated with certain EV sources or isolation strategies. Conceptually relevant association of size-based EV isolation with protein cargo and uptake function will guide strategic conclusions enhancing future EV research and product development. Based on this study, an open-source database is built to facilitate further analysis with conventional or AI tools to identify additional causative associations of interest.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type–specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFβ and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFβ, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6β4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Background Epidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB). Methods In this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene. Results As analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents. Conclusion Here we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.

Stromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation.

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.