Abstract
INTRODUCTION: Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of mesenchymal stem cells (MSCs) marked by stage-specific embryonic antigen 3 (SSEA3), exhibit superior regenerative capacity compared to conventional MSCs, including enhanced tissue homing, pluripotency, and paracrine effects. However, their natural scarcity (1-5% in MSC populations) limits therapeutic scalability. In this study, we developed a five-compound small molecule method to obtain compound-enriched Muse-like MSCs and assessed their potential use in treating severe veterinary chronic diseases, such as hepatitis and chronic kidney disease (CKD). METHODS: Umbilical cord-derived MSCs from cats and dogs were isolated and cultured, using a combination of five small molecules to obtain enriched Muse-like cells. The cultivation process was verified by immunofluorescence and flow cytometry. Differentiation potential of obtained Muse-like cells was evaluated under lineage-specific conditions. These compound-enriched Muse-like MSCs were administered intravenously (2 × 10(6) cells/kg) to a 6-year-old cat with severe hepatitis (twice a week for 2 weeks) and a 16-year-old dog with CKD (weekly for 4 weeks). Serum biochemistry and clinical observations were monitored pre- and post-treatment. RESULTS: Screening of over 100 small-molecule compounds identified optimized five-compound cocktails-valproic acid (0.5 mM), CHIR99021 (3 μM), PD0325901 (0.5 μM), Trolox (10 μM), and nicotinamide (1 mM) for feline MSCs; parnate (10 μM), CHIR99021 (3 μM), PD0325901 (0.5 μM), Trolox (10 μM), and Y27632 (10 μM) for canine MSCs. These small-molecule cocktail effectively boosted SSEA3 positivity, from 0.1-1% to approximately 40%, as confirmed by immunofluorescence staining and flow cytometry. These enriched Muse-like cells demonstrated superior stress tolerance and robust spontaneous differentiation into endodermal (KRT7 + hepatocyte-like), mesodermal (cTnI+ cardiomyocyte-like), and ectodermal (Nestin+ neural progenitor) lineages under targeted induction conditions, which were not observed in untreated MSCs. In therapeutic applications, the enriched Muse-like MSCs normalized feline liver indices by day 21 and improved canine renal markers by day 28, accompanied by notable anti-aging effects. CONCLUSION: A small molecule cocktail method was introduced to enhance the Muse population in MSCs, which provide a safe and effective way to harvest Muse cells. These Muse-like MSCs demonstrate high clinical potential for chronic and age-related degenerative diseases, making it a safe and effective therapeutic option.