Transfection and transplantation of eNSCs

CEND1-GFP-PURO and control GFP-PURO lentiviruses were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China), with a titer of 2 × 10⁸ transducing units/mL. eNSCs were plated on dishes coated in laminin and poly-L-lysine in a proliferative culture solution. When cell cultures were 80% confluent, they were transfected with CEND1-GFP-PURO or control GFP-PURO lentiviruses; transfection efficiency was improved by the addition of puromycin (Sigma) to the culture medium.

Next, eNSCs were transplanted into the peri-injury regions at Target Point 1 (post bregma = –0.5 mm, lateral to the midline = 3 mm, depth = 1.5 mm), Target Point 2 (post bregma = –2 mm, lateral to the midline = 4.5 mm, depth = 1.5 mm), and Target Point 3 (post bregma = –3.5 mm, lateral to the midline = 3 mm, depth = 1.5 mm) at 3 days after TBI. Subsequently, an injection of PBS, eNSC control (eNSC transfected with control GFP-PURO lentiviruses), or CEND1-eNSCs (eNSC transfected with the CEND1-GFP-PURO lentiviruses) (3 × 10⁵ cells in 3 μL PBS) was made into each target point at a rate of 1 μL/min using a Hamilton syringe and micro-infusion pump (WPI, Sarasota, FL, USA) attached to a stereotaxic device (Stoelting, Kiel, WI, USA).