Human Stem Cell Transplantation, Immunosuppression, and Noninvasive In vivo Cell Tracking in the Mouse Brain

Stem cell therapies have emerged as potential therapeutics of interest for many central nervous system diseases, including stroke, epilepsy, neurodegenerative diseases, and traumatic brain injury. Preclinical models are required to evaluate the migration of transplanted stem cells to various areas of the brain, monitor graft survival to improve immunosuppression regimens, and assess stem cell safety and their efficacy on disease outcomes. Understanding all these aspects of stem cells as therapeutics can help optimize parameters for translation to humans. To facilitate this overarching goal, we report a protocol for human stem cell transplantation into mouse fimbria fornix, the outflow white matter tract of the hippocampus, which facilitates stem cell dissemination to regions distant to the injection site. Although our protocol involves injecting human neural stem cells expressing insulin-like growth factor 1 into an Alzheimer's disease mouse model, it can be altered for application to diverse stem cells and cellular therapeutics, secreting various trophic factors, at multiple dosing schemes, and in different central nervous system targets and disorder models. Our approach is robust and highly reproducible, with demonstrated success in improving memory performance in an Alzheimer's disease mouse model and achieving long-term survival of transplanted cells (up to 32 weeks) in immunocompetent mice, facilitating stem cell studies in the context of neuroimmune crosstalk. Our primary protocol involves administering an anti-CD4/anti-CD40L monoclonal antibody cocktail to prevent graft rejection, with bioluminescence imaging to track transplanted stem cells in vivo. We also present an alternate immunosuppression protocol that relies on tacrolimus and mycophenolate mofetil, which is less costly, albeit less effective (especially in the longer term), than the CD4-CD40L antibody regimen. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Human stem cell transplantation into mouse hippocampus, antibody-based immunosuppression, and in vivo tracking by bioluminescence imaging Alternate Protocol: Immunosuppression by tacrolimus and mycophenolate mofetil.