Comprehensive Preclinical Safety Evaluation of Clinical-Grade hESC-Derived Multipotent Mesenchymal Stem Cells for Interstitial Cystitis.

Abstract

Human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (MMSCs) offer therapeutic potential for interstitial cystitis (IC); however, safety concerns, such as tumorigenicity from residual undifferentiated hESCs, must be thoroughly addressed. To address these concerns, we conducted comprehensive in vitro and in vivo safety assessments of clinical-grade MMSCs. Flow cytometry, qRT-PCR, and/or RNA sequencing confirmed the absence of pluripotency markers, including OCT3/4, TRA-1-60, TRA-1-81, and NANOG, in MMSCs. In good laboratory practice (GLP)-compliant studies, BALB/c-nu/nu immunodeficient mice received a single intra-detrusor muscle injection of MMSCs. No adverse clinical signs or immune responses were observed at doses corresponding to up to 6 × 10⁹ cells in humans, the maximum feasible clinical dose based on body weight conversion. Furthermore, no tumor formation was detected for up to 52 weeks following either intra-detrusor muscle or subcutaneous administration. Spiking studies showed teratoma formation only when undifferentiated hESCs exceeded 1%, supporting the absence of tumorigenic risk in the final MMSC product. Biodistribution analyses revealed that MMSCs remained localized at the injection site and were cleared from major organs within 10 days. These findings indicate that MMSCs contain no detectable hESCs within the sensitivity limits of the assays, exhibit no immunogenicity or tumorigenic potential, and are safe for intra-detrusor muscle administration, supporting their clinical application as a cell-based therapy for IC.