Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model

•Nucleus-restricted fluorescent labeling of Schwann cells pre-transplantation is described.•Conventional GFP labeling technique can result in underestimation of survival counts.•Nuclear labeling offers precise approach of quantifying Schwann cell survival in vivo.•Presence of NRG1β in the injectate media enhances Schwann cell survival in vivo. To optimize survival evaluation of Schwann cells (SCs) in vivo, we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 β (NRG1β) improves in vivo survival. We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1β in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14μm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed. Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2). We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome. Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1β in the injectate media can improve survival.