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Stem cell research & therapy2025; 16(1); 547; doi: 10.1186/s13287-025-04671-1

Generation of equine induced pluripotent stem cells from cells of embryonic, perinatal and adult tissues.

Abstract: Regenerative therapies are quickly expanding to application in equine patients because of their importance as sporting and companion animals. Furthermore, aligning with a One Health concept, veterinary medicine offers a unique platform for preclinical studies. While mesenchymal stem/stromal cells (MSCs) therapies are already used in treating horses, strategies involving induced pluripotent stem cells (iPSCs) are poorly developed. iPSCs present great potential for therapy and disease modelling, but their consistent generation in horses requires further investigation into the source of somatic cells and the reprogramming method and conditions. Methods: The reprogramming potential of equine cells from tissues of three developmental origins was compared: prenatal (embryo-derived MSCs, eMSCs), perinatal (cord blood-derived MSCs, CB-MSCs) and adult (articular chondrocytes, ACs). Two reprogramming methods (retroviral, lentiviral) and different culture conditions (serum/serum-free, feeder cells/feeder-free, with/without small molecules) were tested. Pluripotent gene expression was analyzed at different time-points to reveal transcriptomic changes associated with reprogramming. The generated equine iPSCs (eqiPSCs) were characterized by alkaline phosphatase (AP) staining, expression of pluripotent genes and proteins, three-germ layer differentiation (embryoid body) and karyotype. Results: Using a lentiviral vector with serum-free media and feeder cells resulted in the most favorable conditions for eqiPSCs reprogramming, but adding small molecules had a negative effect. Equine CB-MSCs and ACs were only partially reprogrammed and could not be efficiently expanded in culture. Only eMSCs generated putative eqiPSCs that met the cellular, molecular and functional criteria of pluripotent cells. Equine eMSCs showed higher proliferation and basal expression of pluripotent genes compared to CB-MSCs and ACs, and showed the highest upregulation of pluripotent genes along reprogramming. Conclusions: The developmental stage of the starting cell strongly influences their reprogramming potential in equine species. This has been suggested for human and other animal species, but direct comparison of equine cells from prenatal, perinatal and adult sources has not been reported before. Novel preliminary insight into the transcriptomic changes of different equine cell types during reprogramming, and on the effect of different culture conditions, can contribute improving the generation of eqiPSCs. While transgene-free methods are the goal, putative eqiPSCs are critical to enlarge our knowledge on animal iPSC biology.
© 2025. The Author(s).
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Publication Date: 2025-10-08 PubMed ID: 41063189PubMed Central: PMC12506261DOI: 10.1186/s13287-025-04671-1Google Scholar: Lookup
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  • Journal Article

Summary

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Overview

  • This study explores the generation of induced pluripotent stem cells (iPSCs) from different equine tissue sources at various developmental stages, evaluating the best methods and conditions for efficient reprogramming.
  • The research aims to improve the development of equine iPSCs (eqiPSCs) for potential regenerative therapies, disease modeling, and preclinical veterinary applications.

Introduction and Background

  • Equine patients, valuable both as sporting and companion animals, are increasingly benefiting from regenerative medicine approaches.
  • Veterinary medicine offers a unique “One Health” platform to conduct preclinical studies that may translate across species, including humans.
  • While mesenchymal stem/stromal cells (MSCs) derived therapies are already in use for horses, the generation and utilization of equine induced pluripotent stem cells (eqiPSCs) remain underdeveloped.
  • iPSCs possess characteristics of pluripotency — the ability to differentiate into any cell type — making them highly promising for therapeutic and disease modeling applications.

Research Objective

  • To systematically compare the reprogramming potential of equine cells derived from three developmental stages: prenatal (embryonic mesenchymal stem cells, eMSCs), perinatal (cord blood MSCs, CB-MSCs), and adult (articular chondrocytes, ACs).
  • To evaluate two distinct viral reprogramming methods (retroviral and lentiviral) and varying culture conditions affecting iPSC generation.
  • To analyze gene expression and cellular characteristics at multiple time points to identify transcriptomic changes during reprogramming.

Methods

  • Cell Sources: Equine cells from three distinct origins:
    • Embryo-derived MSCs (eMSCs) — prenatal stage
    • Cord blood MSCs (CB-MSCs) — perinatal stage
    • Articular chondrocytes (ACs) — adult stage
  • Reprogramming Techniques: Two viral vector systems employed for delivering pluripotency genes:
    • Retroviral vectors
    • Lentiviral vectors
  • Culture Conditions Tested:
    • Serum-containing versus serum-free media
    • Feeder cells presence versus feeder-free culture
    • Use of small molecules (bioactive compounds that can affect reprogramming efficiency) versus no additives
  • Characterization Techniques:
    • Alkaline phosphatase (AP) staining to identify pluripotent cells
    • Quantitative gene expression analysis for pluripotency markers at multiple time points
    • Protein expression analysis to confirm pluripotency
    • Functional tests with embryoid body differentiation to demonstrate capacity to form all three germ layers
    • Karyotyping to assess chromosomal stability of generated eqiPSCs

Results

  • Optimal Reprogramming Conditions:
    • Lentiviral vectors combined with serum-free media and feeder cells provided the most efficient reprogramming environment.
    • The inclusion of small molecules surprisingly had a negative effect on reprogramming efficiency in this context.
  • Cell Source Impact:
    • Cord blood MSCs (CB-MSCs) and adult articular chondrocytes (ACs) were only partially reprogrammed and lacked capacity for successful expansion of pluripotent cells.
    • Embryo-derived MSCs (eMSCs) uniquely generated putative eqiPSCs that satisfied established criteria for pluripotent stem cells at the molecular, cellular, and functional level.
    • eMSCs demonstrated higher baseline proliferation and endogenous expression of pluripotent genes compared to CB-MSCs and ACs, correlating with superior reprogramming success.
    • During reprogramming, eMSCs showed the most significant upregulation of genes associated with pluripotency.

Conclusions and Significance

  • The developmental origin of the starting somatic cells significantly affects reprogramming outcome in horses — prenatal cells have higher reprogramming potential than perinatal or adult cells.
  • This finding supports observations from human and other species but uniquely provides direct comparative data for equine cells from multiple developmental stages.
  • Insights into transcriptomic changes across cell types during reprogramming and the influence of culture conditions can guide the optimization of eqiPSC generation protocols.
  • Though non-integrating transgene-free reprogramming methods are the ultimate aim for clinical applications, establishing putative eqiPSCs through current methods is vital for advancing basic understanding of equine iPSC biology.
  • These advancements will aid in developing regenerative therapies for horses and strengthen veterinary medicine’s role in One Health translational research.

Cite This Article

APA
Barrachina L, Ivanovska A, Eslami Arshaghi T, O'Brien A, Cequier A, Murphy M, Hollinshead F, Rodellar C, Barry F. (2025). Generation of equine induced pluripotent stem cells from cells of embryonic, perinatal and adult tissues. Stem Cell Res Ther, 16(1), 547. https://doi.org/10.1186/s13287-025-04671-1

Publication

Stem cell research & therapy
ISSN: 1757-6512
NlmUniqueID: 101527581
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 547
PII: 547

Researcher Affiliations

Barrachina, Laura
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
  • Laboratorio de Genética Bioquímica (LAGENBIO), Universidad de Zaragoza; Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA; Instituto de Investigación Sanitaria de Aragón (IIS), Universidad de Zaragoza, Zaragoza, Spain.
Ivanovska, Ana
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
Eslami Arshaghi, Tarlan
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
O'Brien, Aisling
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
Cequier, Alina
  • Laboratorio de Genética Bioquímica (LAGENBIO), Universidad de Zaragoza; Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA; Instituto de Investigación Sanitaria de Aragón (IIS), Universidad de Zaragoza, Zaragoza, Spain.
Murphy, Mary
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
Hollinshead, Fiona
  • Animal Reproduction and Biotechnology Laboratory (ARBL), Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
Rodellar, Clementina
  • Laboratorio de Genética Bioquímica (LAGENBIO), Universidad de Zaragoza; Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA; Instituto de Investigación Sanitaria de Aragón (IIS), Universidad de Zaragoza, Zaragoza, Spain.
Barry, Frank
  • Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland. frank.barry@universityofgalway.ie.

MeSH Terms

  • Animals
  • Horses
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / metabolism
  • Cellular Reprogramming
  • Cell Differentiation
  • Cells, Cultured

Grant Funding

  • 101026825 / European Union's Horizon 2020, Marie Sklodowska-Curie Actions
  • PID2020-116352GB-I00 / Ministerio de Ciencia e Innovación (Spain)

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: Equine eMSCs were isolated at Colorado State University (Colorado, US) under ethics approval of the Institutional Animal Care and Use Committee (title: “Production of equine fetally-derived mesenchymal stem cells and isolation and characterization of their extracellular vesicles”; approval number: 4443; approval date: 17 April 2023). Equine cord blood was obtained under informed owner consent at Irish stud farms, and CB-MSCs were isolated at University of Galway (Galway, Ireland). Ethical approval was not required because the blood was collected from peripartum waste material by non-invasive means for the mare or the foal. Equine BM-MSCs were obtained at the University of Zaragoza (Zaragoza, Spain) under ethics approval of the in-house Advisory Ethics Committee for Animal Research (title: “Optimización del uso de MSCs alogénicas en el tratamiento de patologías articulares equinas: equilibrio inmunomodulación-inmunogenicidad” [“Optimizing the use of allogenic MSCs for treating equine joint pathologies: immunomodulation-immunogenicity balance”]; approval number: PI 15/16; approval date: 22 June 2021). Equine ACs were obtained post mortem at the University of Liverpool (UK) from healthy joints of animals euthanized for reasons unrelated to this study, thus ethics approval was not required but informed owner’s consent was obtained. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

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