Assessment of the safety of hypoxia-primed mesenchymal stem cells derived from umbilical cord and adipose tissues in animals

Mesenchymal stem cells (MSCs) are widely used for disease treatment, typically cultured in ambient oxygen (21%) exceeding physiological levels (2-5%). While hypoxic MSCs show in vitro advantages, their in vivo effects remain less understood. This study evaluated the safety of xeno-free, serum-free cultured MSCs from adipose tissue (AD-MSCs) and umbilical cords (UC-MSCs) primed with 5% oxygen in healthy rabbits, Swiss mice, and rats. Assessments included vascular and muscle stimulation, systemic hypersensitivity, acute toxicity, and subchronic toxicity. Our data indicated that the injection of UC-MSCs induced vascular stimulation but not muscular stimulation, whereas the injection of AD-MSCs caused neither vascular nor muscular stimulation. Additionally, neither AD-MSCs nor UC-MSCs affected hematopoietic parameters (white blood cell, red blood cell, platelet, or hemoglobin levels), altered the levels of proinflammatory cytokines (IL-6, IL-1β, IFN-γ, and TNF-a), or induced allergenic responses in rabbits. Furthermore, intravenous injection of either UC-MSCs or AD-MSCs at a dose of 50 × 10⁶ cells/kg did not cause acute toxicity in mice. However, injections of higher doses of these cells led to intravenous thrombosis and embolism in various organs of experimental animals, ultimately resulting in animal death. Finally, according to the subchronic toxicity assay, the administration of MSCs generally did not impact liver, kidney, or spleen function in rats. In conclusion, hypoxic AD-MSCs and UC-MSCs are safe for potential therapeutic use with consideration of thrombogenic risk. We suggest that these hypoxic MSC lines could be alternatives to normoxic MSCs for disease treatment.