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Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)