Tra 1 60

The main reagents include DMEM/F12 (1:1) medium, knockout serum replacement (KSR), valproic acid, fetal bovine serum, Essential 8™ Flex Medium, Geltrex™, KSFM medium, BMP‐4, bovine pituitary extract (BPE), (Gibco), NANOG, OCT4, SOX2, SSEA‐4, TRA‐1‐81, TRA‐1‐60, Krt19, Integrinβ1, CD200, CD31 and VEGF‐A antibodies (Abcam), PDGF‐B and Ang2 antibodies (Santa Cruz), recombinant human EGF (R&D), RA, valproic acid, sodium alginate (Sigma), Matrigel^® Matrix (Corning), mTeSRTM1 medium (Stem Cell), Astragalus polysaccharide (Solarbio), silk fibroin and collagen (Hefei Bomei Bio), and Dextran Texas red™ (Invitrogen). Primers were designed using Primier 6.0 software, and all primers were synthesized as shown in Table 1.

The cultured cells were placed on 12 mm cover slips, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min at room temperature, washed three times with phosphate-buffered saline (PBS, Geno, China), and treated with a permeabilizing and blocking buffer (10% donkey serum, 0.225% Triton X-100) for 1 hour at room temperature. Then, the cells were incubated with the following primary antibodies: OCT4 (1 : 200, Abcam), Nanog (1 : 250, Abcam), SSEA4 (1 : 250, Abcam), TRA-1-60 (1 : 300, Abcam), SOX1 (1 : 300, Boster), SOX2 (1 : 300, Boster), Nestin (1 : 300, Abcam), Olig2 (1 : 500, Millipore), Pax6 (1 : 100, DSHB), HB9 (1 : 50, DSHB), Islet1 (1 : 250, Abcam), ChAT (1 : 100, Millipore), and TuJ1 (1 : 250, Abcam). All antibodies were diluted in antibody dilution buffer (2% donkey serum, 0.05%Triton X-100), and the cells were incubated with the antibodies overnight at 4°C. After three washing steps, the cells were incubated for 1 hour at room temperature with secondary antibodies: Alexa Fluor (488 or 555) donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat. For the detection of AChR, the cocultured cells were incubated with Alexa Fluor 555-conjugated α-BTX (1 μg/ml, Invitrogen) for 1 hour at 37°C before fixation. The cells were then washed 2 times in PBS and fixed with 4% PFA. All cell samples were observed using an Olympus fluorescence microscope.

For immunofluorescence, frozen sections of chondrocyte spheres were washed with PBS for three times, these cell slices were permeabilized with cold 0.2% Triton X-100 (Sigma, USA) in PBS for 5 min. A step of enzymatic antigen retrieval with 0.1% Trypsin in PBS was performed prior to block for 1 h. After blocking, antibodies against Aggrecan (1:300; Abcam, UK), COL II (1:300; Abcam), SOX9 (1:200; Affinity), MMP13 (1:200; Affinity), COL X (1:500; Abcam), NANOG (1:300; Abcam), OCT4 (1:300; Abcam), TRA-1-60(1:300; Abcam) and Ki67 (1:300; Abcam) were added overnight. The following day, cells were washed three times with PBS and then incubated at 37 °C for 1 h with Alexa 594-conjugated goat anti-rabbit secondary antibody (1:300; Abcam) or Alexa 488-conjugated goat anti-mouse secondary antibody (1:300; Abcam). The cell nucleus was counterstained with DAPI (Beyotime, People’s Republic of China).

Cells grown on 10% HSt-PMEDSAH-g dishes were fixed in 4% paraformaldehyde for 10 min at room temperature followed by permeabilized with 0.1% Triton X-100 for 10 min. Primary antibodies raised against OCT3/4 (Cell signaling), SOX2 (MilliporeSigma, Burlin-tong, MA, USA), NANOG (Abcam, Cambridge, UK), TRA-1-60 (Abcam), and TRA-1-81 (MilliporeSigma) were diluted in 1% normal donkey serum and incubated overnight at 4 °C with gentle shaking and detected with respective secondary antibodies. Micrographs were captured using an EVOS®FL Cell Imaging System (Thermo Fisher Scientific).