Direct reprogramming of human fetal retinal pigment epithelial cells into photoreceptor-like cells [RNA-seq]

RNAseq profiles for the CiPCs induction 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.

本数据集包含CiPCs诱导实验的RNA测序转录组谱：将胎儿视网膜色素上皮（fetal retinal pigment epithelium, fRPE）细胞以3×10⁴ 个细胞/平方厘米的密度接种于0.1%明胶包被的培养板，于第0天（D0）采用fRPE培养基进行培养。次日（D1），将培养基更换为感光细胞诱导培养基1（PIM1），该培养基包含DMEM/F12、10% knockout血清替代物、2% B27添加剂、5 ng/mL骨形态发生蛋白抑制剂Noggin、5 ng/mL胰岛素样生长因子1（IGF-1），并联合添加5种小分子化合物（5F）：0.5 mM丙戊酸（VPA）、4.8 μM CHIR99021、2 μM Repsox、10 μM毛喉素（Forskolin）及10 μM IWR1。为促进并维持感光细胞的形成，于D5至D10期间，在含有5F的培养基（PIM2）中添加3 nM音猬因子（sonic hedgehog）、100 μM牛磺酸、1 μM视黄酸（合称STR）、1% N2添加剂及5 ng/mL碱性成纤维细胞生长因子（bFGF）。针对人真皮成纤维细胞（human dermal fibroblast, HDF）重编程实验，将细胞接种于生长培养基中。于D1加入含5F的PIM1（浓度与前述一致），并于D5更换为含STR组合与5F的PIM2培养基。关于PTBP1反义寡核苷酸（antisense oligonucleotide, ASO）处理实验：于D0接种细胞，使用生长培养基培养，直至24小时后细胞汇合度达到70%~80%。于D1，在更换为含5F的PIM1培养基的同时，使用Lipofectamine RNAimax转染试剂（赛默飞世尔科技，ThermoFisher Scientific）进行转染，转染体系为：每1 mL培养基中添加3.75 μL RNAimax与37.5 pmol ASO。反义寡核苷酸处理48小时后，更换为含5F的新鲜PIM1培养基，后续操作与前述流程一致。