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

Overview

• This study compared mesenchymal stromal cells (MSCs) derived from equine ovarian follicular aspirates to dermal fibroblasts to evaluate how well standard identification methods distinguish between these two closely related cell types.
• The research analyzed differences in cell morphology, expansion, surface markers, differentiation capacity, and gene expression profiles to find markers unique to MSCs versus fibroblasts.

Background and Rationale

• MSCs are multipotent cells widely used for regenerative therapies due to their ability to differentiate into multiple cell types and modulate immune responses.
• Equine MSCs are being explored for use in veterinary regenerative medicine.
• MSCs have been isolated from equine ovarian follicular aspirates, providing a novel source of these cells.
• A major challenge is fibroblast contamination during MSC isolation because fibroblasts share many characteristics with MSCs, including spindle-like shape and certain surface markers.
• The International Society for Cellular Therapy (ISCT) minimal criteria for MSCs (such as specific surface markers and trilineage differentiation potential) are sometimes insufficient to distinguish MSCs from fibroblasts conclusively.

Study Objectives

• To compare morphological characteristics and growth behavior of MSCs derived from equine follicular aspirates to a commercial equine dermal fibroblast cell line.
• To assess the immunophenotype by measuring expression of surface markers associated with MSCs and hematopoietic cells.
• To evaluate differentiation capacity into the three typical mesenchymal lineages (adipogenic, osteogenic, chondrogenic).
• To analyze differences in gene expression profiles between the two cell types using RT² Profiler PCR Array and qPCR validation.

Methods

• Cell culture under standard conditions was performed for both follicular aspirate-derived MSCs and dermal fibroblasts.
• Morphology was observed to identify spindle-like shape typical for both cell types.
• Immunophenotyping assessed markers such as CD90, CD29 (positive for MSCs), and CD45, CD19, MHC-II (negative for MSCs).
• Trilineage differentiation assays were performed to measure adipogenic, osteogenic, and chondrogenic potential.
• Transcriptomic profiling was conducted using a PCR array focused on MSC-related genes, followed by quantitative PCR validation of selected differentially expressed genes.

Key Findings

• Both MSCs and fibroblasts displayed typical spindle-like morphology.
• Both expressed mesenchymal markers (CD90, CD29) and lacked hematopoietic markers (CD45, CD19) and MHC-II expression, meeting ISCT MSC criteria.
• Both cell types could differentiate into adipogenic and osteogenic lineages, but chondrogenic differentiation was less pronounced in fibroblasts.
• Transcriptomic analysis identified 35 genes differentially expressed between the two cell types.
• Fibroblasts had higher expression of ANPEP, PROM1, and SOX2; while MSCs showed higher levels of FGF2, LIF, KDR, and notably GDF6, which is important for MSC differentiation.
• These molecular differences suggest some genes may serve as distinguishing markers beyond conventional surface markers and differentiation assays.

Conclusions and Implications

• Standard criteria for identifying MSCs (morphology, surface markers, trilineage differentiation) are insufficient to reliably distinguish MSCs from fibroblasts due to significant overlap.
• Transcriptomic profiling reveals distinct gene expression patterns that can potentially discriminate MSCs from fibroblasts more accurately.
• This research highlights the need to incorporate additional molecular or functional assays when isolating and validating MSC populations, especially from tissue sources susceptible to fibroblast contamination.
• The findings can improve the purity and characterization of MSC preparations for equine regenerative medicine, potentially enhancing their therapeutic efficacy and safety.