Derivation of genetically stable mesenchymal stem cells from feline embryonic cell aggregates

Mesenchymal stem cells (MSCs) are regarded as a fundamental component in the advancement of regenerative therapies for feline medicine. In this study, a novel protocol was established to derive MSCs from discarded feline embryos obtained post-ovariohysterectomy. Embryonic cell aggregates (EAs) were generated through collagenase digestion of embryos, followed by three-dimensional (3D) culture in complete medium containing 100 ng/mL basic fibroblast growth factor (bFGF). The EAs were subsequently plated on collagen-coated dishes to yield feline embryonic cell aggregate-derived MSCs (feEA-MSCs). Strong expression of characteristic MSCs markers CD73, CD90, and CD105 was demonstrated by surface marker analysis, with minimal expression of CD34 and HLA-DR. Furthermore, significantly higher mRNA expression of the pluripotency-associated transcription factors NANOG and SOX2 was exhibited by feEA-MSCs compared to adipose-derived MSCs (AD-MSCs) (P < 0.05). Consistent alkaline phosphatase activity was maintained by the cells, while a karyotypically normal state at passage 6 (2n = 38) was retained. Overall, this approach provides a source of feline MSCs with enhanced pluripotency characteristics, demonstrating significant potential for translational applications in regenerative medicine.