Comparative phenotypic and transcriptomic analysis of equine ovarian follicular aspirates-derived mesenchymal stromal cells and dermal fibroblasts

Mesenchymal stromal cells (MSCs) are widely used in human cell-based therapies and recent research is focused on the use of MSCs in equine regenerative medicine. Recently, MSCs have been isolated from equine follicular aspirates; however, a major concern during isolation is the potential contamination with fibroblasts, the predominant stromal cell type in many tissues. Furthermore, both cell types share the minimal criteria established by the International Society for Cellular Therapy (ISCT) for MSCs identification. Hence, our study aimed to compare the morphological characteristics, expansion behavior, immunophenotype, differentiation capacity and gene expression profiles of equine MSCs obtained from follicular aspirates and a commercial equine dermal fibroblast cell line. Under standard culture conditions, both cell lines exhibited a spindle-like morphology, expressed mesenchymal markers such as CD90 or CD29, and lacked the expression of CD45, CD19 and MHC-II, and manifested trilineage differentiation, however the chondrogenic differentiation was less evident in fibroblasts. Transcriptomic analysis using an RT² Profiler PCR Array revealed 35 differentially expressed genes between MSCs and fibroblasts. These results were further validated by quantitative PCR analysis: while fibroblasts showed higher expression of ANPEP, PROM1 and SOX2, genes like FGF2, LIF and KDR were downregulated in these cells. Moreover, GDF6, a key factor for MSCs differentiation, was markedly downregulated in fibroblasts. In conclusion, our results demonstrate that conventional criteria, including morphology, immunophenotyping, differentiation assays, and gene expression analysis, are insufficient to unequivocally identify MSCs. These findings underscore the need to implement additional experimental approaches to confidently discriminate between MSCs and fibroblasts.