Rats were sacrificed with an overdose of pentobarbital sodium (100 mg/kg, intraperitoneal injection) and the NPCs, AFCs and DCs were prepared as previously described (15 (link),17 (link),18 (link)). The cells used in this experiment were at the third passage (P3). The cells type and purity were identified by immunofluorescence staining for collagen-II and aggrecan. The cells were blocked with 5% donkey serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min at room temperature and incubated overnight with rabbit anti-rat collagen II (1:200; cat. no. ab188570; Abcam, Cambridge, MA, USA) and rabbit anti-rat aggrecan (1:200, cat. no. 13880-1-AP, ProteinTech Group, Inc., Chicago, IL, USA) primary antibodies at 4°C. Subsequently, following an incubation with goat anti-rat secondary antibody (1:500, cat. no. M21003, Abmart, Inc., Shanghai, China) for 2 h at room temperature, nuclei were stained DAPI. The observation was carried out with a fluorescence microscope (Olympus Corp., Tokyo, Japan). Transplanted NPCs expressing GFP (GFP-NPCs) were isolated from GFP transgenic SD rats and used to identify the location of the transplanted cells.