Isolation of SSEA-3-Positive Muse Cells in Canine and Feline Adipose Tissues

Abstract

Muse cells are endogenous pluripotent-like stem cells identified as stage-specific embryonic antigen-3 (SSEA-3)-positive subpopulations in the bone marrow, peripheral blood, and connective tissues of various organs. Clinical trials conducted by intravenous injection of donor-Muse cells, without the use of immunosuppressive drugs, have demonstrated safety and efficacy across multiple diseases. Since the epitope recognized by the anti-SSEA-3 antibody is a glycolipid, rather than a protein produced by a genetic code, the antibody may detect Muse cells across different species. Muse cells possess unique properties, including the ability to survive under stressful conditions, spontaneously turn into different cell types from all three primary layers of the body, and repair tissues in living organisms. They have been isolated from several mammalian species. However, their presence and characteristics in companion animals, such as canine and feline, remain unexplored, despite the growing demand for treatments that regenerate tissues in veterinary medicine. Adipose-derived stem cells (ADSCs) were established from adipose tissue taken during routine veterinary procedures. SSEA-3-positive cells were isolated using fluorescence-activated cell sorting. SSEA-3-positive cells were found in both canine (0.93 ± 0.16%) and feline (2.9 ± 0.15%) ADSCs, similar to human rates. Gene expression analysis revealed that SSEA-3-positive cells exhibited significantly higher levels of the pluripotency markers Oct3/4 and NANOG compared with SSEA-3-negative ADSCs. In suspension culture, SSEA-3-positive cells formed ES cell-like M-clusters. These cells could differentiate into endodermal (SOX17, AFP), mesodermal (GATA2, DESMIN, SMA), and ectodermal (NESTIN, NF) marker-positive cells, as measured by quantitative polymerase chain reaction and immunocytochemistry. These results show that canine and feline ADSCs contain SSEA-3-positive cells. These cells express pluripotency markers and can differentiate into endodermal, mesodermal, and ectodermal lineages. Their properties match those of Muse cells in humans and other mammals. This study offers basic evidence for isolating Muse cells from pets and demonstrates their potential for use in veterinary regenerative therapies.