Effects of continuous passage on the immunomodulatory properties of equine bone marrow-derived mesenchymal stem cells in vitro.
Abstract: The immunomodulatory properties of mesenchymal stem cells (MSCs) have been studied extensively due to their increasing clinical application for tissue regeneration and repair following culture expansion. We have studied the effect of continuous passage on the immunomodulatory capacity of equine bone marrow-derived MSCs (BM-MSCs). Equine BM-MSCs were isolated and culture expanded to passage three, six, and nine (P3, P6, P9). Immunomodulatory properties of each passage were assessed using a T cell proliferation assay and cytokine synthesis following stimulation with interferon gamma (IFN-γ). Results: Equine BM-MSCs maintained their primary cell morphology and immunophenotype throughout all passages. T cell proliferation was suppressed by all passages of BM-MSCs, compared to peripheral blood mononuclear cells (PBMCs) alone. There was no significant difference in suppression of T cell proliferation between P3, P6, and P9 BM-MSCs. All passages of BM-MSCs significantly increased cytokine synthesis in response to stimulation with IFN-γ. There were no significant differences in production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) or regulate on activation, normal T cell expressed and secreted (RANTES) following stimulation with IFN-γ between P3, P6, and P9 BM-MSCs. P9 BM-MSCs had significantly increased production of tumor necrosis factor alpha (TNF-α), (IL-1β), and (IL-10) compared to P3 BM-MSCs. Additionally, there was a significant increase in production of (IL-8) in P6 and P9 BM-MSCs in comparison to P3 BM-MSCs. Conclusions: Our findings demonstrate that culture expansion affects some of the immunomodulatory properties of BM-MSCs in vitro, which may suggest that MSCs isolated from a single collection of bone marrow may be culture expanded, but only those from lower passage numbers would be ideal for clinical application.
Copyright © 2021 Elsevier B.V. All rights reserved.
Publication Date: 2021-02-12 PubMed ID: 33636546DOI: 10.1016/j.vetimm.2021.110203Google Scholar: Lookup
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- Journal Article
Summary
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This research investigates how the continuous culture of equine bone marrow-derived mesenchymal stem cells (BM-MSCs) affects their ability to modulate immune responses. The results showed that although the BM-MSCs maintained their ability to suppress T cell proliferation and related immune functions, the production of some important immune response markers changed significantly in cells cultured over longer periods.
Methodology
- Equine bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated and expanded in culture up to three different stages, referenced as P3, P6, and P9.
- The researchers assessed the cells’ immunomodulatory properties at each stage using a T cell proliferation test and by examining cytokine synthesis after stimulation with interferon gamma (IFN-γ).
Outcomes
- The BM-MSCs maintained their original cell shape and immunophenotype across all passages, indicating they retained their core characteristics over time.
- All the cultures of BM-MSCs suppressed T cell proliferation as compared to peripheral blood mononuclear cells (PBMCs) used as a control. This confirms the immunosuppressive qualities of the MSCs.
- No significant difference was noted in the suppression of T cell proliferation between P3, P6, and P9, suggesting this function does not significantly degrade with passage.
- However, the researchers found that the BM-MSCs’ response to IFN-γ stimulation in terms of cytokine production did change with passage.
Differences in Cytokine Production
- No significant differences were detected in the production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), or the regulate on activation, normal T cell expressed and secreted (RANTES) protein with IFN-γ stimulation.
- However, the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) saw significantly increased production in P9 BM-MSCs compared to those at P3.
- Interleukin-8 (IL-8) also saw increased production in cells at P6 and P9 compared to those at P3.
Conclusions
- The immunomodulatory properties of equine BM-MSCs do change with culture expansion. Specifically, the expression of certain cytokines shifts over time.
- This could mean that only BM-MSCs from early culture stages are ideal for clinical use, as they have more predictable and possibly more beneficial cytokine responses.
Cite This Article
APA
Connard SS, Linardi RL, Even KM, Berglund AK, Schnabel LV, Ortved KF.
(2021).
Effects of continuous passage on the immunomodulatory properties of equine bone marrow-derived mesenchymal stem cells in vitro.
Vet Immunol Immunopathol, 234, 110203.
https://doi.org/10.1016/j.vetimm.2021.110203 Publication
Researcher Affiliations
- Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States.
- Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States.
- Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States.
- Department of Clinical Sciences, College of Veterinary Medicine and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States.
- Department of Clinical Sciences, College of Veterinary Medicine and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States.
- Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: kortved@vet.upenn.edu.
MeSH Terms
- Animals
- Bone Marrow Cells / drug effects
- Bone Marrow Cells / immunology
- Bone Marrow Cells / physiology
- Cell Differentiation / drug effects
- Cell Differentiation / immunology
- Cell Proliferation / drug effects
- Cells, Cultured
- Cytokines / analysis
- Cytokines / biosynthesis
- Cytokines / immunology
- Female
- Horses
- Immunomodulation
- In Vitro Techniques
- Interferon-gamma / pharmacology
- Lymphocyte Activation / drug effects
- Lymphocyte Activation / immunology
- Male
- Mesenchymal Stem Cells / immunology
- Mesenchymal Stem Cells / physiology
Citations
This article has been cited 8 times.- Cequier A, Vázquez FJ, Romero A, Vitoria A, Bernad E, García-Martínez M, Gascón I, Barrachina L, Rodellar C. The immunomodulation-immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility. Front Vet Sci 2022;9:957153.
- Even KM, Gaesser AM, Ciamillo SA, Linardi RL, Ortved KF. Comparing the immunomodulatory properties of equine BM-MSCs culture expanded in autologous platelet lysate, pooled platelet lysate, equine serum and fetal bovine serum supplemented culture media. Front Vet Sci 2022;9:958724.
- Deng J, Li D, Huang X, Li W, Zhao F, Gu C, Shen L, Cao S, Ren Z, Zuo Z, Deng J, Yu S. Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway. Mol Biol Rep 2022 Sep;49(9):8337-8347.
- Kiselevskii MV, Vlasenko RY, Stepanyan NG, Shubina IZ, Sitdikova SM, Kirgizov KI, Varfolomeeva SR. Secretome of Mesenchymal Bone Marrow Stem Cells: Is It Immunosuppressive or Proinflammatory?. Bull Exp Biol Med 2021 Dec;172(2):250-253.
- Liu S, Peng H, Meng J, Zhuo L, Fu S, Yang W. Cell passage number drives transcriptomic drift as an overlooked factor in experimental reproducibility. Sci Rep 2025 Nov 21;15(1):44984.
- Vachkova E, Arnhold S, Petrova V, Heimann M, Koynarski T, Simeonova G, Piperkov P. Transcriptional Factors Related to Cellular Kinetics, Apoptosis, and Tumorigenicity in Equine Adipose-Derived Mesenchymal Stem Cells (ASCs) Are Influenced by the Age of the Donors. Animals (Basel) 2025 Jun 28;15(13).
- Reynolds DE, Vallapureddy P, Morales RT, Oh D, Pan M, Chintapula U, Linardi RL, Gaesser AM, Ortved K, Ko J. Equine mesenchymal stem cell derived extracellular vesicle immunopathology biomarker discovery. J Extracell Biol 2023 Jun;2(6):e89.
- Maged G, Abdelsamed MA, Wang H, Lotfy A. The potency of mesenchymal stem/stromal cells: does donor sex matter?. Stem Cell Res Ther 2024 Apr 22;15(1):112.
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