Recombinant human ngf

Recombinant human NGF (R&D Systems), SEMA3A (R&D Systems), and human SLIT1 (R&D Systems), diluted in Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies) were used as candidate agents with a potential to affect the axonal growth of hESC- and hiPSC-derived RGCs. DPBS was used as the control. These were added to RMM at final concentrations of 50, 100, or 200 ng/ml for NGF and SEMA3A, and 0.2, 1, 2, or 5 μg/ml for SLIT1. Further, SEMA3A and SLIT1 were supplemented on D27, at the time of attachment. For the supplementation assay of NGF, the induction protocol of RGCs was slightly modified, because the previously established protocol induced maximal growth of RGC axons. In particular, the supplementation of NGF was initiated on D24 without FBS or retinoic acid. On D27, attachment of floating EBs was achieved in the absence of FBS or BDNF^{26 (link)}. Supplementation of these agents was continued until D30, 3 days after attachment, and the colonies were collected on D30.

PC12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% heat inactivated horse serum, 5% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and 100 U/ml penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37°C, 5% CO2, and saturated humidity. PC12 cells were differentiated in the DMEM containing 1% heat inactivated horse serum and 100 U/ml penicillin-streptomycin. Cells were treated with recombinant human NGF (R&D, USA) at the concentration of 50 ng/ml for 7 days. ThePD model was induced by incubation of differentiated PC12 cells with 800 μM of MPP+ (Sigma-Aldrich, MO, USA) for 24 hours.