Neurobasal b27

Synthetic Humanin (HN) and unrelated peptide (UP) were purchased from Sangon biotech (Shanghai China). The sequences of HN and UP are MAPRGFSCLLLLTSEIDLPVKRRA and IYMCILTVYPAEAISQWGRDLAVD, respectively. The Neurobasal/B27 and DMEM/F-12 were purchased from Gibco-BRL (Grand Island, NY, USA). Poly-D-lysine (MW 150,000–300,000), trypsin, arabinoside cytosine, Calcein-AM, N-Acetyl-l-cysteine (NAC), NMDA, and MK-801 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan), and Kit of LDH was from Njjcbio (Nanjing, China). Fluo-3AM and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) were from Invitrogen (Carlsbad, California, USA). MAPK and Phospho-MAPK Family Antibody Sampler Kits were obtained from Cell Signaling Technology (Beverly, MA, USA). β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). New born Wistar rats were from the Experimental Animal Center of Shanxi Medical University.

A schematic diagram of the protocol for induced pluripotent stem cell (iPSC) differentiation to retinal ganglion cells is shown in Figure 1a. The confluent iPSCs were scraped off into small aggregates and transferred to non-adherent dishes containing an MTeSR^TM1 medium (STEMCELL Technologies, Vancouver, BC, Canada), where they were cultivated for 10 days to generate embryoid bodies (EBs). On the tenth day (Day 10), EBs were transferred to plates coated with 0.1% gelatin and were cultivated in hES medium supplemented with 10% FBS for seven days until neural rosettes appeared. Following protocol, the optic vesicles (OVs) could be obtained at Day 18. The OVs were then stimulated to differentiate into RGCs by culturing for one day in DMEM/F12 containing an N-2 supplement (Gibco, Carisbad, CA, USA), on the following day the medium was replaced with Neurobasal medium/B27 (Gibco, Carisbad, CA, USA) supplemented with DAPT, and on the fifth day the medium was replaced with Neurobasal/B27. On the 15th day (Day 25), the differentiated RGCs could be observed.

The female SD rats were selected to be 18 days pregnant. The uterus was removed by laparotomy, the placenta was removed, and the spinal cord of the fetal rat was quickly separated and temporarily stored in a 0°C Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA). The spinal blood vessel and the membrane were removed under the stereomicroscope. The spinal cord was put into a 15 mL centrifuge tube in which Trypsin (Gibco, USA) was added for digestion at 37°C for 15 min. DMEM‐containing serum was then added to stop digestion. The cells were filtered using a 200 mesh cell screen and centrifuged at 1200 rpm for 5 min. The supernatant was removed and the planting medium (DMEM + 20% FBS, Gibco, USA) was added again. After cell counting, the cells were inoculated into polylysine‐coated culture plates at a density of 106 cells/mL, and cultured at 37°C with 5% CO₂. Four hours later, the medium was replaced with a special culture medium for neuron growth (neurobasal+B27, Gibco, USA). Cytarabine (2.5 μg/mL) was added to the medium, and half of the solution was replaced with culture for 3 days to inhibit the growth of glial cells and obtain the purest primary neurons. The neurons were cultured until the seventh day, and identified by immunofluorescence assay with specific mark antibody β3‐tubulin.