Neural graft elimination via dual safety switch compromises therapeutic recovery after stroke

Safety switch systems are increasingly incorporated into stem cell therapies to enable selective graft elimination in the event of adverse outcomes. Yet how removing transplanted cells affects the underlying pathology remains largely unexplored. Here, we show that iPSC-derived neural stem cells (NSCs) co-transduced with herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase-9 (iC9) are more efficiently ablated by combined ganciclovir (GCV) and chemical inducer of dimerization (CID) treatment than either single switch alone, under both proliferating and differentiating conditions. Using bioluminescence imaging to track graft survival longitudinally, we demonstrate that dual safety switch NSC (DS-NSC) grafts are selectively eliminated by GCV and CID administration in immunodeficient mice following photothrombotic stroke. The combination treatment initiated on day 8 post-transplantation resulted in reduced graft volume, lower NSC proliferation, and persistently reduced bioluminescence signal compared to mice receiving only solvent. Histological analysis revealed that combination treatment was associated with larger stroke lesions, higher IBA1 fluorescence intensity, and reduced vascular density in the ischemic border zone. Mice receiving GCV and CID showed persistently elevated contralateral hindlimb error rates on a ladder walk task from day 28 post-stroke onward compared to solvent-treated mice. These findings demonstrate that while DS-NSC elimination is feasible, the pharmacological activation of the safety switch system is associated with impaired functional stroke recovery.