Isolation and characterization of mesenchymal stromal cells derived from equine ovarian follicular aspirates.

Overview

• This study demonstrates that mesenchymal stromal cells (MSCs) can be isolated from equine ovarian follicular aspirates collected during ovum pick-up (OPU) procedures in mares.
• The isolated cells were characterized, expanded, and successfully differentiated into multiple lineages, suggesting a new source of MSCs for equine reproductive and regenerative therapies.

Research Context and Objective

• MSCs are increasingly explored for therapeutic use in equine medicine, especially for reproductive pathologies.
• In human medicine, MSCs have been isolated from follicular aspirates collected during transvaginal oocyte aspiration for reproductive treatments, providing a novel autologous cell source.
• Despite the common use of transvaginal oocyte aspiration (OPU) to collect equine oocytes for in vitro embryo production, this method had not been evaluated as a source of MSCs in mares prior to this study.
• The goal was to assess whether follicular aspirates from mares contain viable MSCs that can be isolated, characterized, and expanded for potential therapeutic applications.

Methodology

• Follicular aspirates were obtained from six mares through the OPU process, which is a minimally invasive transvaginal technique to collect oocytes.
• The aspirates were processed to establish adherent cell cultures, allowing MSCs to grow in vitro.
• Cell morphology was examined, and surface protein expression was analyzed using flow cytometry with antibodies targeting:
  • MSC markers: CD29 and CD90 (positive indicators)
  • Hematopoietic markers: CD45 and CD19 (negative indicators)
  • Major histocompatibility complex class II (MHC-II), also expected to be negative in MSCs
• Gene expression analysis was performed by RT-qPCR to evaluate MSC-related gene markers:
  • THY1 and FGF2 are genes associated with MSC characteristics and were specifically monitored.
  • Comparisons were made between native follicular aspirates and cultured MSCs to confirm enrichment.
• Functional multipotency was assessed by inducing differentiation of cultured cells into three mesenchymal lineages:
  • Adipogenic (fat)
  • Osteogenic (bone)
  • Chondrogenic (cartilage)

  Specific staining methods and morphological changes were used to confirm lineage differentiation.

Key Findings

• The isolated cells displayed spindle-like morphology, a characteristic feature of MSCs.
• Flow cytometry confirmed that the cells expressed MSC surface markers (CD29 and CD90) and did not express hematopoietic or immune markers (CD45, CD19, MHC-II), indicating purity of the MSC population.
• RT-qPCR showed significant upregulation of THY1 and FGF2 genes in cultured cells compared to native aspirates, demonstrating selective expansion and enrichment of MSCs during culture.
• The cells exhibited trilineage differentiation capacity, successfully differentiating into adipocytes, osteocytes, and chondrocytes as confirmed by specific staining techniques.

Implications and Applications

• The study identifies ovarian follicular aspirates in horses as a new, practical, and minimally invasive source of MSCs.
• This source could facilitate autologous cell therapies in equine reproduction, aiding treatment of ovarian or reproductive disorders.
• Beyond reproduction, MSCs derived from follicular aspirates might be used in broader regenerative medicine applications for horses.
• The technique leverages an already established OPU procedure, integrating cell collection with routine oocyte retrieval without extra invasive steps.
• This approach offers an alternative to other MSC sources, such as bone marrow or adipose tissue, potentially reducing animal discomfort and simplifying cell procurement.

Conclusion

• Equine ovarian follicular aspirates contain a viable and multipotent MSC population that can be isolated and expanded efficiently in vitro.
• The findings open up new avenues for cell-based therapies in equine reproductive medicine and tissue regeneration, utilizing cells derived from ovarian follicular fluids obtained during routine oocyte collection.