Phenotypic changes associated with continuous long term in vitro expansion of bone marrow-derived mesenchymal stem...

In vitro expansion of mesenchymal stem cells is necessary to obtain a higher cell number for clinical applications. However, long-term expansion can produce significant phenotypic changes on these cells, decreasing their therapeutic utility. Therefore, understanding the phenotypic changes that long-term expansion triggers in mesenchymal stem cells will allow for better and more consistent cell therapy results. Here, we evaluate the phenotypic changes caused by continuous passaging through colony forming unit-fibroblast assay, senescence beta-galactosidase staining, morphology examination, secretome analysis, surface marker expression, protein quantification, osteogenic and adipogenic differentiation, and CD4⁺ T lymphocyte immunosuppressive potential. Long-term in vitro culture decreases mesenchymal stem cell osteogenic potential and self-renewal, increases cell size, and senescence, but does not consistently affect adipogenic differentiation. Surface marker expression remains similar for positive and negative markers, while secretory phenotype shifts with decreased p14ARF, MMP-3, p21 Waf1/Cip1,ENA-78, GCP-2, GROα, IL-3, IL-7, IL-8, RANTES, TNFβ, and VEGF-A expression, and increased p53, p16 INK4a, MCP-1, and SDF-1 expression. Immunomodulatory potential remains unchanged. These findings can help better understand the phenotypic changes that mesenchymal stem cells undergo while expanded in vitro.