GSE192816
Direct reprogramming of human fetal retinal pigment epithelial cells into photoreceptor-like cells [Methyl-seq]
DNA methylation profiles for the  CiPCs induction Overall design: fRPE cells were plated on 0.1% gelatin coated plates at a density of 3 x 104 cells/cm2 and cultured in fRPE medium on Day 0 (D0). The next Day (D1), medium was changed to photoreceptor induction medium 1 (PIM1) containing DMEM/F12, 10% knockout serum replacement, 2% B27, 5 ng/mL Noggin, 5 ng/mL IGF-1, in combination with 5 small molecule factors (5F): 0.5 mM VPA, 4.8 µM CHIR99021, 2 µM Repsox, 10 µM Forskolin and 10 µM IWR1. To promote and support the formation of photoreceptors, 3 nM sonic hedgehog, 100 µM taurine, 1 µM retinoic acid (STR), 1% N2 and 5 ng/mL bFGF were added in the medium (PIM2) along with 5F from D5 to D10.  For HDF reprogramming, cells were seeded in growth medium. On D1, PIM1 containing 5F (at the same concentrations) was added, and PIM2 containing STR and 5F was added on D5. For PTBP1 ASO treatment, Cells were seeded on D0 and cultured in growth medium to reach a confluency at around 70%-80% 24 hours later. On D1, in addition to medium change of PIM1 with 5F, PTBP1 ASO was transfected with Lipofectamine RNAimax (ThermoFisher Scientific). We used 3.75 µL of RNAimax, 37.5 pmol ASO in 1 ml of culture medium. 48 hours after ASO treatment, medium was replaced by fresh PIM1 containing 5F and the following process was the same as described above.