Effects of continuous passage on the immunomodulatory properties of equine bone marrow-derived mesenchymal stem cells in vitro.

Background: 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.
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