SCI and spinal cord-derived NSPC culture

All animal experiments in this study were carried out in accordance with the National Institute of Health guide for the care and use of Laboratory animals (NIH Publications No. 80-23) revised 1996, approved by the Medical Ethics Committee at Xijing Hospital of the Fourth Military Medical University, and all efforts were made to minimize animal suffering and reduce animals used.

Briefly, three 6-week-old female adult C57/BL6 mice were anesthetized with 5% isoflurane and subjected to laminectomy at the T9–10 vertebral level. Contusion SCI was induced with an NYU impactor by dropping a 5-g rod 6.25 mm above the spinal cord. After skin closure, mice were placed on a warming pad until they were completely awake. Manual bladder emptying was done twice a day for 3 days. The mice with SCI were anesthetized again, and their T6–T12 spinal cords were isolated, placed in 10-cm dishes containing cold PBS, dissociated, and filtered through a 70-μm strainer (BD Falcon). The cells were plated at a density of 1 × 10⁶ cells per mL in NSPC growth media (Dulbecco’s modified Eagle medium, DMEM/F12, B-27, N-2, Gibco) and passaged every 7 days (Fig. ​(Fig.11A).

Schematic illustration of sp-NSPC culture, differentiation, and experimental design. A sp-NSPCs, harvested from injured spinal cords, differentiated into neurons, astrocytes, and oligodendrocytes. B Transplantation of LINGO-1 shRNA-treated sp-NSPCs and timeline for motor function analysis