Itaconate is a metabolic regulator of bone formation in homeostasis and arthritis.

OBJECTIVES: Bone remodelling is a highly dynamic process dependent on the precise coordination of osteoblasts and haematopoietic-cell derived osteoclasts. Changes in core metabolic pathways during osteoclastogenesis, however, are largely unexplored and it is unknown whether and how these processes are involved in bone homeostasis. METHODS: We metabolically and transcriptionally profiled cells during osteoclast and osteoblast generation. Individual gene expression was characterised by quantitative PCR and western blot. Osteoblast function was assessed by Alizarin red staining. immunoresponsive gene 1 (Irg1)-deficient mice were used in various inflammatory or non-inflammatory models of bone loss. Tissue gene expression was analysed by RNA in situ hybridisation. RESULTS: We show that during differentiation preosteoclasts rearrange their tricarboxylic acid cycle, a process crucially depending on both glucose and glutamine. This rearrangement is characterised by the induction of Irg1 and production of itaconate, which accumulates intracellularly and extracellularly. While the IRG1-itaconate axis is dispensable for osteoclast generation in vitro and in vivo, we demonstrate that itaconate stimulates osteoblasts by accelerating osteogenic differentiation in both human and murine cells. This enhanced osteogenic differentiation is accompanied by reduced proliferation and altered metabolism. Additionally, supplementation of itaconate increases bone formation by boosting osteoblast activity in mice. Conversely, Irg1-deficient mice exhibit decreased bone mass and have reduced osteoproliferative lesions in experimental arthritis. CONCLUSION: In summary, we identify itaconate, generated as a result of the metabolic rewiring during osteoclast differentiation, as a previously unrecognised regulator of osteoblasts.

Analysis of extracellular vesicle microRNA profiles reveals distinct blood and lymphatic endothelial cell origins.

Extracellular vesicles (EVs) are crucial mediators of cell-to-cell communication in physiological and pathological conditions. Specifically, EVs released from the vasculature into blood were found to be quantitatively and qualitatively different in diseases compared to healthy states. However, our understanding of EVs derived from the lymphatic system is still scarce. In this study, we compared the mRNA and microRNA (miRNA) expression in blood vascular (BEC) and lymphatic (LEC) endothelial cells. After characterization of the EVs by fluorescence-triggered flow cytometry, nanoparticle tracking analysis and cryo-transmission electron microscopy (cryo-TEM) we utilized small RNA-sequencing to characterize miRNA signatures in the EVs and identify cell-type specific miRNAs in BEC and LEC. We found miRNAs specifically enriched in BEC and LEC on the cellular as well as the extracellular vesicle level. Our data provide a solid basis for further functional in vitro and in vivo studies addressing the role of EVs in the blood and lymphatic vasculature.

The Multifaceted Actions of PVP-Curcumin for Treating Infections.

Curcumin is a natural compound that is considered safe and may have potential health benefits; however, its poor stability and water insolubility limit its therapeutic applications. Different strategies aim to increase its water solubility. Here, we tested the compound PVP-curcumin as a photosensitizer for antimicrobial photodynamic therapy (aPDT) as well as its potential to act as an adjuvant in antibiotic drug therapy. Gram-negative E. coli K12 and Gram-positive S. capitis were subjected to aPDT using various PVP-curcumin concentrations (1-200 µg/mL) and 475 nm blue light (7.5-45 J/cm2). Additionally, results were compared to aPDT using 415 nm blue light. Gene expression of recA and umuC were analyzed via RT-qPCR to assess effects on the bacterial SOS response. Further, the potentiation of Ciprofloxacin by PVP-curcumin was investigated, as well as its potential to prevent the emergence of antibiotic resistance. Both bacterial strains were efficiently reduced when irradiated with 415 nm blue light (2.2 J/cm2) and 10 µg/mL curcumin. Using 475 nm blue light, bacterial reduction followed a biphasic effect with higher efficacy in S. capitis compared to E. coli K12. PVP-curcumin decreased recA expression but had limited effect regarding enhancing antibiotic treatment or impeding resistance development. PVP-curcumin demonstrated effectiveness as a photosensitizer against both Gram-positive and Gram-negative bacteria but did not modulate the bacterial SOS response.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Elimination of damaged mitochondria during UVB-induced senescence is orchestrated by NIX-dependent mitophagy.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

Aged-vascular niche hinders osteogenesis of mesenchymal stem cells through paracrine repression of Wnt-axis.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Profiling microRNA expression during senescence and aging: mining for a diagnostic tool of senescent-cell burden.

In the last decade cellular senescence, a hallmark of aging, has come into focus for pharmacologically targeting aging processes. Senolytics are one of these interventive strategies that have advanced into clinical trials, creating an unmet need for minimally invasive biomarkers of senescent cell load to identify patients at need for senotherapy. We created a landscape of miRNA and mRNA expression in five human cell types induced to senescence in-vitro and provide proof-of-principle evidence that miRNA expression can track senescence burden dynamically in-vivo using transgenic p21 high senescent cell clearance in HFD fed mice. Finally, we profiled miRNA expression in seven different tissues, total plasma, and plasma derived EVs of young and 25 months old mice. In a systematic analysis, we identified 22 candidate senomiRs with potential to serve as circulating biomarkers of senescence not only in rodents, but also in upcoming human clinical senolytic trials.

Week-by-week changes in serum levels of bone-related circulating microRNAs and bone turnover markers.

MicroRNAs are involved in post-transcriptional regulation of gene expression. Due to their regulatory role, microRNAs are differently expressed during specific conditions in healthy and diseased individuals, so microRNAs circulating in the blood could be used as diagnostic and prognostic biomarkers for various diseases and conditions. We want to investigate the variability of circulating microRNAs and bone turnover markers in weekly time intervals in older women. In a single-site longitudinal study, a panel of 19 bone-related miRNAs was measured using the osteomiR RT-qPCR assay in serum samples of 35 postmenopausal women divided into 3 groups: healthy controls (n = 12), low BMD (n = 14), and vertebral fractures (n = 9). Blood samples for measurement of CTX, PINP, OC, and bone ALP were collected once per week for 8 weeks at 9:00 AM after overnight fasting. Serum samples from all participants were analyzed for 19 microRNA bone biomarkers and 4 bone turnover markers over 8 weeks. We analyzed the data using a mixed model analysis of variance and found no significant changes between week-by-week time points in any of the groups. To estimate intraindividual variability between weekly time points, we have calculated the median coefficient of variation (CV). This was between 28.4% and 80.2% for microRNA, with an assay CV of 21.3%. It was between 8.5% and 15.6% for bone turnover markers, with an assay CV of 3.5% to 6.5%. The intraindividual variability was similar between groups. Circulating microRNAs measured in serum had a higher weekly intraindividual variability than bone turnover markers due in part to a higher assay CV.

Vesicular Messages from Dental Biofilms for Neutrophils.

The encounter between dental biofilm and neutrophils in periodontitis remains elusive, although it apparently plays a crucial role in the periodontal pathology and constitutes a key topic of periodontology. Dental biofilm and neutrophils were isolated from orally healthy persons and patients with periodontitis. We investigated biofilm and its particle-shedding phenomenon with electron microscopy and nanoparticle tracking analysis (NTA); biofilm shedding-neutrophil interactions were examined ex vivo with epi-fluorescence microscopy. For this purpose, we used acellular dental biofilm shedding, purified lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) as activators, and the interleukin 8 receptor beta (CXCR2) inhibitor and the anti-interleukin 8 receptor alpha (CXCR1) antibody as modulators. The shedding of acellular dental biofilms overwhelmingly consists of bacterial extracellular vesicles (BEVs). The latter induced the moderate formation of neutrophil extracellular traps (NETs) in orally healthy subjects and a strong formation in patients with periodontitis. A CXCR2 inhibitor and an anti-CXCR1 antibody had a minor effect on NET formation. Neutrophils from patients with periodontitis exhibited NET hyper-responsiveness. BEVs were stronger inducers of NET formation than purified LPS and PMA. A plateau of neutrophil responsiveness is reached above the age of 40 years, indicating the abrupt switch of maladaptive trained immunity (TI) into the activated modus. Our results suggest that dental biofilms consist of and disseminate immense amounts of outer membrane vesicles (OMVs), which initiate NET formation via a non-canonical cytosolic LPS/caspase-4/11/Gasdermin D pathway. This modus of NET formation is independent of neutrophil elastase (NE), myeloperoxidase (MPO), peptidylarginine deiminase 4 (PAD4), and toll-like receptors (TLR). In periodontitis, the hyper-responsiveness of neutrophils to BEVs and the increased NET formation appear to be a consequence of TI.

Prospective Study on the Influence of Occupational Hand Protection Products on the Efficacy of Hand Disinfection.

BACKGROUND: To prevent occupational skin diseases, employees are instructed to periodically apply hand protection products as a barrier to protect their hands from water, cleaning agents or other irritants. The aim of this work was to investigate whether bacteria present on the skin at the time of protection product application are enclosed underneath this protective layer, if they can be transferred to other surfaces and if a standard isopropanol-based skin disinfectant can nonetheless reduce the bacterial burden. METHODS: This prospective study was conducted in human volunteers based on the European Standard (EN 1500) to assess the burden of microorganisms before and after the application of various protection product formulations and subsequent hand disinfection. RESULTS: All protection products, with the exception of alcohol-based gels, enclosed bacteria underneath a lipid layer which could be transferred onto other surfaces. Still, the hand disinfectant efficiently reduced the bacteria burden. DISCUSSION: In occupations where proper hand hygiene is vital, alcohol-based gels might be the best option for the protection of the skin barrier as well as for reducing the contamination risk. CONCLUSION: An alcohol-based disinfection agent can dissolve the lipid film of protection products following the standard protocol for hygienic hand disinfection.

2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

Current senolytics: Mode of action, efficacy and limitations, and their future.

Senescence is a cellular state characterized by its near-permanent halted cell cycle and distinct secretory phenotype. Although senescent cells have a variety of beneficial physiological functions, progressive accumulation of these cells due to aging or other conditions has been widely shown to provoke deleterious effects on the normal functioning of the same or higher-level biological organizations. Recently, erasing senescent cells in vivo, using senolytics, could ameliorate diseases identified with an elevated number of senescent cells. Since then, researchers have struggled to develop new senolytics each with different selectivity and potency. In this review, we have gathered and classified the proposed senolytics and discussed their mechanisms of action. Moreover, we highlight the heterogeneity of senolytics regarding their effect sizes, and cell type specificity as well as comment on the exploited strategies to improve these features. Finally, we suggest some prospective routes for the novel methods for ablation of senescent cells.

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients.

Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.

Circulating microRNAs in young individuals with long-duration type 1 diabetes in comparison with healthy controls.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional control of gene expression and might be used as biomarkers for diabetes-related complications. The aim of this case-control study was to explore potential differences in circulating miRNAs in young individuals with long-duration type 1 diabetes (T1D) compared to healthy controls, and how identified miRNAs are expressed across different tissues. Twelve adolescents, age 15.0-17.9 years, with T1D duration of more than 8 years (mean 11.1 years), were enrolled from the Swedish diabetes quality registry. An age-matched control group was recruited. Circulating miRNAs (n = 187) were analyzed by quantitative PCR. We observed that 27 miRNAs were upregulated and one was downregulated in T1D. Six of these miRNAs were tissue-enriched (blood cells, gastrointestinal, nerve, and thyroid tissues). Six miRNAs with the largest difference in plasma, five up-regulated (hsa-miR-101-3p, hsa-miR-135a-5p, hsa-miR-143-3p, hsa-miR-223-3p and hsa-miR-410-3p (novel for T1D)) and one down-regulated (hsa-miR-495-3p), with P-values below 0.01, were selected for further in-silico analyses. AKT1, VEGFA and IGF-1 were identified as common targets. In conclusion, 28 of the investigated miRNAs were differently regulated in long-duration T1D in comparison with controls. Several associations with cancer were found for the six miRNAs with the largest difference in plasma.

Mesenchymal Stem Cell Conditioned Medium Modulates Inflammation in Tenocytes: Complete Conditioned Medium Has Superior Therapeutic Efficacy than Its Extracellular Vesicle Fraction.

Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.

Senopathies-Diseases Associated with Cellular Senescence.

Cellular senescence describes a stable cell cycle arrest state with a characteristic phenotype. Senescent cells accumulate in the human body during normal aging, limiting the lifespan and promoting aging-related, but also several non-related, pathologies. We propose to refer to all diseases whose pathogenesis or progression is associated with cellular senescence as "senopathies". Targeting senescent cells with senolytics or senomorphics is likely to mitigate these pathologies. Examples of senopathies include cardiovascular, metabolic, musculoskeletal, liver, kidney, and lung diseases and neurodegeneration. For all these pathologies, animal studies provide clear mechanistic evidence for a connection between senescent cell accumulation and disease progression. The major persisting challenge in developing novel senotherapies is the heterogeneity of senescence phenotypes, causing a lack of universal biomarkers and difficulties in discriminating senescent from non-senescent cells.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

PRPF19 modulates morphology and growth behavior in a cell culture model of human skin.

The skin provides one of the most visual aging transformations in humans, and premature aging as a consequence of oxidative stress and DNA damage is a frequently seen effect. Cells of the human skin are continuously exposed to endogenous and exogenous DNA damaging factors, which can cause DNA damage in all phases of the cell cycle. Increased levels of DNA damage and/or defective DNA repair can, therefore, accelerate the aging process and/or lead to age-related diseases like cancer. It is not yet clear if enhanced activity of DNA repair factors could increase the life or health span of human skin cells. In previous studies, we identified and characterized the human senescence evasion factor (SNEV)/pre-mRNA-processing factor (PRPF) 19 as a multitalented protein involved in mRNA splicing, DNA repair pathways and lifespan regulation. Here, we show that overexpression of PRPF19 in human dermal fibroblasts leads to a morphological change, reminiscent of juvenile, papillary fibroblasts, despite simultaneous expression of senescence markers. Moreover, conditioned media of this subpopulation showed a positive effect on keratinocyte repopulation of wounded areas. Taken together, these findings indicate that PRPF19 promotes cell viability and slows down the aging process in human skin.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.