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Stem cell research & therapy2025; 16(1); 162; doi: 10.1186/s13287-025-04287-5

Determination of the miRNA profile of extracellular vesicles from equine mesenchymal stem cells after different treatments.

Abstract: Background: Osteoarthritis (OA) is a common and incurable disease in humans and animals. To gain a better understanding of the pathogenesis and identify potential treatments, miRNAs will be extracted and analysed from extracellular vesicles (EVs) of equine adipose derived mesenchymal stem cells (AdMSCs).Methods: For this purpose we cultivated and pretreated AdMSCs under different conditions: interleukin 1β, shock wave, chondrogenic differentiation, chondrogenic differentiation under hypoxia, or after senescence. After treatment, EVs were harvested from the cell culture supernatants. Next-generation sequencing (NGS) was used to sequence the miRNAs from the EVs.Results: A total of 89 miRNAs whose expression was significantly altered compared with that of an untreated negative control were identified. On average, 53 miRNAs were upregulated and 6 miRNAs were downregulated. Among others, the miRNAs eca-miR-101, eca-miR-143, eca-miR-145, eca-miR-146a, eca-miR-27a, eca-miR-29b, eca-miR-93, eca-miR-98, and eca-miR-221 were significantly increased after the stimulations, which, as known anti-inflammatory miRNAs, could be candidates for therapeutic use in the treatment of OA.Conclusion: These results lay the foundation for further research into the significance and efficacy of these miRNAs so that this knowledge can be improved in further experiments and, ideally, translated into therapeutic use.
© 2025. The Author(s).
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Publication Date: 2025-04-05 PubMed ID: 40188160PubMed Central: PMC11972531DOI: 10.1186/s13287-025-04287-5Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study examined which microRNAs inside extracellular vesicles released by horse fat-derived stem cells change after different lab stimulations, aiming to pinpoint anti-inflammatory candidates for osteoarthritis therapy. They found 89 significantly altered microRNAs—mostly increased—including several known to dampen inflammation, laying groundwork for future therapeutic development.

What the study set out to do

  • Profile the microRNA (miRNA) cargo of extracellular vesicles (EVs) secreted by equine adipose-derived mesenchymal stem cells (AdMSCs) after distinct biological stimulations or states.
  • Identify miRNAs whose abundance changes in EVs and that could contribute to anti-inflammatory or cartilage-protective effects relevant to osteoarthritis (OA).

Why EV miRNAs matter for osteoarthritis

  • EVs are nano-sized particles naturally released by cells that deliver regulatory molecules, including miRNAs, to other cells and tissues.
  • miRNAs fine-tune gene expression; in joints, they can influence inflammation, cartilage matrix turnover, and cell survival—key processes in OA.
  • Engineering or selecting EVs enriched in therapeutic miRNAs is a promising, cell-free strategy that may reduce risks associated with administering live cells.

Experimental design and treatments

  • Source cells: Equine AdMSCs (fat-derived mesenchymal stem cells) cultured under standard conditions.
  • Pretreatments applied to AdMSCs to model different joint-relevant contexts:
    • Interleukin-1β (IL-1β): mimics inflammatory milieu of OA.
    • Shock wave stimulation: models a common orthopedic biophysical therapy that can modulate MSC secretomes.
    • Chondrogenic differentiation: pushes MSCs toward a cartilage-like phenotype.
    • Chondrogenic differentiation under hypoxia: reflects low-oxygen cartilage environment, which can alter MSC/EV biology.
    • Senescence: models aged or stress-exposed cells often present in chronic joint disease.
  • After each condition, EVs were collected from culture supernatants for miRNA analysis.

miRNA profiling approach

  • Next-generation sequencing (NGS) was used to comprehensively sequence small RNAs within EVs.
  • Differential expression was determined by comparing each treatment group to an untreated control.
  • Significance criteria were applied to identify miRNAs with altered abundance; details such as fold-change thresholds, multiple-testing correction, and replicate numbers are not provided in the abstract and would be important for full interpretation.

Key findings

  • Total of 89 EV-associated miRNAs showed significant changes versus untreated controls across the tested conditions.
  • On average per condition, 53 miRNAs were upregulated and 6 were downregulated, indicating a strong tendency toward increased EV miRNA cargo after stimulation.
  • Several miRNAs known in the literature for anti-inflammatory or joint-protective roles were significantly increased after stimulations, including:
    • eca-miR-101, eca-miR-143, eca-miR-145, eca-miR-146a
    • eca-miR-27a, eca-miR-29b
    • eca-miR-93, eca-miR-98, eca-miR-221
  • These candidates are highlighted as potentially therapeutic for OA.

Biological interpretation and relevance to OA

  • Anti-inflammatory signaling:
    • miR-146a is widely associated with negative feedback on inflammatory pathways and could temper cytokine-driven cartilage catabolism.
    • miR-101, miR-93, miR-98, and miR-221 have been implicated in modulating cytokine production, NF-κB activity, or immune-cell responses in various systems.
  • Matrix remodeling and chondrogenesis:
    • miR-29b and miR-27a are linked to extracellular matrix regulation and may influence collagen and proteoglycan turnover relevant to cartilage integrity.
    • miR-143 and miR-145 are associated with mesenchymal lineage regulation and could affect chondrocyte differentiation and phenotype stability.
  • Context dependence:
    • The pattern and magnitude of miRNA changes likely differ by stimulus (inflammation, hypoxia, mechanical shock, differentiation, senescence), enabling tailored EV profiles for specific therapeutic goals.
    • Hypoxia and chondrogenic cues may enrich EVs in miRNAs that support cartilage matrix production, while IL-1β may induce anti-inflammatory miRNAs as a cellular counter-response.

What makes this study useful

  • Establishes an equine-specific EV miRNA catalog under clinically relevant stimuli, a valuable resource for translational OA research in veterinary and human medicine.
  • Identifies concrete miRNA candidates for further functional validation and therapeutic EV engineering.
  • Demonstrates that MSC pretreatment is a lever to tune EV cargo composition toward desired biological effects.

Important limitations and caveats

  • Details not specified in the abstract:
    • EV isolation and characterization methods (e.g., size-exclusion, ultracentrifugation, marker profiling) that influence purity and miRNA content.
    • Number of biological replicates, sequencing depth, normalization strategy, and statistical thresholds (including multiple-testing corrections).
    • Whether changes reflect per-EV cargo alterations versus differences in EV release rates.
  • NGS identifies association, not function:
    • Increased abundance does not guarantee a therapeutic effect; functional assays are required.
    • miRNA effects are context- and dose-dependent, and many miRNAs have pleiotropic targets.
  • Species-specificity:
    • Equine miRNA nomenclature (eca-) and targets may not map one-to-one to human orthologs; cross-species translation must be empirically tested.

Implications and next steps

  • Analytical validation:
    • Confirm NGS results with targeted qRT-PCR for the highlighted miRNAs across independent donors and batches.
    • Quantify EV number and size (e.g., nanoparticle tracking analysis) to distinguish cargo changes from EV release changes.
  • Functional testing:
    • Apply EVs from each pretreatment to inflamed equine chondrocytes or synoviocytes and measure cytokines, MMPs/ADAMTS, and matrix synthesis.
    • Use antagomirs/mimics to test causal roles of specific miRNAs within EVs.
    • Assess joint-protective effects in relevant in vivo OA models (e.g., equine induced OA), with dose–response and safety readouts.
  • EV engineering and manufacturing:
    • Optimize pretreatment conditions to reproducibly enrich therapeutic miRNAs while minimizing pro-senescent or pro-fibrotic signals.
    • Develop scalable, GMP-aligned EV production and purification with robust potency assays.
  • Mechanistic studies:
    • Map predicted targets and pathways of the candidate miRNAs in equine joint cells; integrate with transcriptomics/proteomics after EV treatment.
    • Determine EV uptake kinetics and joint tissue distribution in vivo.

How specific pretreatments may shape EV cargo (rationale)

  • IL-1β:
    • Triggers inflammatory signaling; cells may package counter-regulatory miRNAs (e.g., miR-146a) into EVs as a homeostatic response.
  • Shock wave:
    • Can activate mechanoresponsive pathways, potentially enhancing secretion and loading of regenerative/anti-inflammatory miRNAs.
  • Chondrogenic differentiation ± hypoxia:
    • Biases cells toward cartilage-supportive programs; hypoxia often stabilizes chondrogenic and anti-catabolic signaling, reflected in EV cargo.
  • Senescence:
    • Alters secretory phenotype; careful assessment is needed because senescence can also elevate pro-inflammatory signals alongside some regulatory miRNAs.

Translational outlook

  • Veterinary relevance:
    • Horses develop naturally occurring OA similar to humans; equine EV therapeutics could improve joint health and performance.
  • Human relevance:
    • Conserved miRNA functions suggest potential translation to human OA, pending cross-species validation and safety profiling.
  • Regulatory path:
    • Requires standardized characterization, potency assays, and demonstration of consistency and mechanism-informed efficacy.

Bottom line

  • The study identifies a set of EV-associated miRNAs—predominantly upregulated by specific MSC pretreatments—with literature support for anti-inflammatory and cartilage-protective roles.
  • These findings provide a focused shortlist for rigorous validation and development of EV-based, cell-free interventions for osteoarthritis.

Cite This Article

APA
Klymiuk MC, Speer J, Marco I, Elashry MI, Heimann M, Wenisch S, Arnhold S. (2025). Determination of the miRNA profile of extracellular vesicles from equine mesenchymal stem cells after different treatments. Stem Cell Res Ther, 16(1), 162. https://doi.org/10.1186/s13287-025-04287-5

Publication

Stem cell research & therapy
ISSN: 1757-6512
NlmUniqueID: 101527581
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 162
PII: 162

Researcher Affiliations

Klymiuk, Michele C
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany. michele.klymiuk@uni-giessen.de.
Speer, Julia
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.
Marco, Isabelle De
  • Clinic of Small Animals, c/o Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.
Elashry, Mohamed I
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.
Heimann, Manuela
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.
Wenisch, Sabine
  • Clinic of Small Animals, c/o Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.
Arnhold, Stefan
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, 35392, Giessen, Germany.

MeSH Terms

  • Animals
  • Mesenchymal Stem Cells / metabolism
  • Mesenchymal Stem Cells / cytology
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Horses
  • Extracellular Vesicles / metabolism
  • Extracellular Vesicles / genetics
  • Cell Differentiation
  • Osteoarthritis / genetics
  • Chondrogenesis / genetics
  • Cells, Cultured

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: The authorization for the tissue collection was obtained at the 16th of May 2018 from the local authorities (Regierungspräsidium Gießen, Wetzlar, Germany) and is registered under the number V 54 − 19 c 20 15 h 02 GI 18/1 kTV 1/2018. There is no special project name for this permission. Consent for publication: Not applicable. Artificial intelligence (AI): The authors declare that they have not use AI-generated work in this manuscript. Competing interests: Not applicable.

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Citations

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    doi: 10.4252/wjsc.v17.i6.109283pubmed: 40585957google scholar: lookup
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