Comparative analysis of mesenchymal stromal cells derived from ovarian follicular aspirates of young and aged mares.

Overview

• This research investigates mesenchymal stromal cells (MSCs) collected from ovarian follicular aspirates of young and aged mares to understand how donor age influences their biological and therapeutic characteristics.

Introduction

• Mesenchymal stromal cells (MSCs) are multipotent cells capable of differentiation into various cell types and are used in cell-based therapies.
• Equine ovarian follicular aspirates are a novel and promising source of MSCs for regenerative medicine applications.
• Age-related variations in MSC properties may impact their clinical effectiveness, necessitating a comparative study between young and aged donor horses (mares).

Study Design and Methods

• Participants: 5 young mares (< 10 years) and 5 aged mares (≥ 18 years).
• MSC Isolation: Cells were isolated from ovarian follicular aspirates, expanded in culture, and cryopreserved at passage 3.
• Post-thaw Assessment: After thawing, several assays were conducted covering:
  • Proliferation (growth rate, cell yield, time to confluence)
  • Immunophenotyping (marker expression via flow cytometry)
  • Differentiation capacity (ability to differentiate into three lineages: adipogenic, chondrogenic, osteogenic)
  • Gene expression profiles (using RT Profiler PCR Array and qPCR validation)

Key Findings – Cellular Characteristics

• Proliferation:
  • No significant difference between young and aged MSCs in terms of proliferation rate, total cell yield, and time to reach 80% confluence.
• Immunophenotyping:
  • Both age groups uniformly expressed MSC-positive markers CD90 and CD29.
  • Neither group expressed hematopoietic or immune cell markers CD45, CD19, or MHC-II, confirming MSC identity.
• Differentiation Capacity:
  • Both young and aged MSCs exhibited qualitative trilineage differentiation (fat, cartilage, bone), suggesting retained multipotency regardless of donor age.

Key Findings – Gene Expression Profiles

• Transcriptomic differences between young and aged MSCs were significant despite similar cell behavior and marker expression.
• MSCs from young mares showed upregulation of:
  • Stemness-related genes (e.g., LIF, POU5F2) important for maintaining undifferentiated state and self-renewal.
  • Genes regulating adipogenic (PPARγ) and chondrogenic (SOX9) differentiation, indicating potential for enhanced lineage commitment.
  • Cell migration-associated markers (MCAM, VCAM), which may influence engraftment and tissue homing.
• MSCs from young mares showed downregulation of:
  • Lineage-specific differentiation genes (e.g., TBX5, SLC2A4), potentially indicating less advanced differentiation.
  • Inflammatory mediators such as IL6, which may reduce pro-inflammatory signaling.
  • Senescence-associated markers (ITGAX, BMP4, SMAD3), suggesting reduced cellular aging and related dysfunction.

Interpretation and Implications

• The comparable proliferation, immunophenotype, and differentiation capacity demonstrate that basic MSC functions remain intact regardless of donor age.
• However, significant transcriptomic alterations reveal that younger MSCs may possess a more “stem-like” state with enhanced regenerative potential and lower inflammatory or senescence signatures.
• These molecular differences could critically affect therapeutic efficacy, such as tissue repair capacity or immunomodulatory functions.
• Age should be considered an important factor when sourcing equine follicular MSCs for clinical applications, as younger donor cells might yield superior outcomes.

Conclusions

• While fundamental MSC characteristics are largely preserved with donor age, transcriptomic profiling reveals distinct molecular changes in aged cells.
• The findings highlight the need for further studies to determine how these gene expression changes impact actual therapeutic performance in vivo.
• This study provides foundational knowledge supporting the preferential use of younger mare-derived follicular MSCs in regenerative therapies and informs future development of equine stem cell banking and treatment protocols.