Differential Gene Expression of Mesenchymal Stromal Cell and Granulosa Cell Markers in Equine Follicular Aspirates.

Overview

• This study investigates the changes in cell types found in equine follicular aspirates during culture, focusing on the selective expansion of mesenchymal stromal cells (MSC) and reduction of granulosa cells (GC) over serial passages in vitro.

Background

• Equine follicular aspirates are samples obtained from ovarian follicles during ovum pick-up procedures.
• These aspirates contain a mixed population of cells, primarily mesenchymal stromal cells (MSC) and granulosa cells (GC).
• MSC are multipotent cells with regenerative properties, whereas GC are specialized somatic cells that surround developing oocytes and play roles in follicle development and hormone production.
• Understanding how to selectively culture and expand MSC while minimizing GC is important for research and therapeutic applications.

Objectives

• To characterize the cellular composition of equine follicular aspirates, focusing on MSC and GC markers.
• To evaluate how in vitro culture and passage of follicular aspirate-derived cells affect the relative abundance of MSC and GC populations.
• To validate whether selective isolation and expansion protocols promote MSC enrichment and reduce GC contamination.

Methods

• Follicular aspirate cells (FAC) were isolated from equine ovarian follicles during ovum pick-up.
• Cells were cultured using a previously validated protocol, passaged up to passage 4 (P4).
• Cell morphology was observed to detect phenotypic changes over passages.
• Gene expression analysis was performed using reverse transcription quantitative PCR (RT-qPCR) targeting mesenchymal/stemness markers and granulosa-cell specific genes.
• Specific genes examined include:
  • Mesenchymal and stemness markers: FN1 (fibronectin 1), THY1 (CD90), FGF2 (fibroblast growth factor 2), CD105 (endoglin)
  • Granulosa cell markers: FSHR (follicle-stimulating hormone receptor), FOXL2 (forkhead box L2), CYP11A1 (cytochrome P450 family 11 subfamily A member 1)

Results

• Freshly isolated FAC showed heterogeneous cell morphologies, indicating a mixed cell population including both MSC and GC.
• After several passages (P3 to P4), cells exhibited a more uniform spindle-shaped morphology consistent with MSC characteristics.
• RT-qPCR revealed a significant increase in the expression of MSC and stemness-associated genes in P4 cells versus freshly isolated FAC, confirming selective MSC expansion and enrichment:
• FN1, THY1, FGF2, and CD105 were markedly upregulated at P4.
• Conversely, GC-specific markers were enriched in the initial freshly isolated FAC population, but their expression sharply decreased in later passages, indicating progressive elimination of granulosa cells:
• FSHR, FOXL2, and CYP11A1 showed significantly higher expression in FAC compared to P4 MSC cultures.

Conclusions

• In vitro culture and serial passage of cells derived from equine follicular aspirates result in selective expansion of mesenchymal stromal cells and reduction of granulosa cell populations.
• The observed spindle-shaped morphology and gene expression profiles at passage 4 confirm enrichment of MSC with stemness properties.
• This selective enrichment protocol is effective for isolating MSC from mixed follicular aspirates, which is valuable for regenerative medicine and reproductive research involving equine cells.
• The progressive loss of granulosa cell-specific gene expression demonstrates that early culture passages can purify MSC populations by eliminating contaminating granulosa cells.