Single-cell RNA-sequencing analysis of human iPSC-derived ocular cells
Single-cell RNA-sequencing analysis of human iPSC-derived ocular cells
iPSCs were seeded at a density of 3,000 cells/well in a six-well plate, 1,000 cells/well in a twelve-well plate, and 500 cells/well in a twenty-four-well plate, containing 500 ng/mL iMatrix-511 silk, and cultured for 10 days. The medium was changed every 2–3 days. For SEAM differentiation, the cells were treated with DM (G-MEM; Thermo Fisher Scientific) supplemented with 10% Knockout Serum Replacement (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 1× MEM non-essential amino acids (Thermo Fisher Scientific), 55 µM monothioglycerol (Wako Pure Chemical), penicillin (Meiji Seika Pharma, Tokyo, Japan), and streptomycin (Meiji Seika Pharma) for 0–28 days; then, the cells were treated with ODM [DM:CnT-Prime without EGF and FGF (CELLnTEC, Bern, Switzerland), 1:1] containing 10 µM Y-27632 (Wako Pure Chemical), 10 ng/mL KGF (Wako Pure Chemical) or EGF for 28–56 days; finally, the cells were treated with OEM (Dulbecco's modified Eagle medium/F12 (Thermo Fisher Scientific)) containing B27-supplement (Thermo Fisher Scientific), 10 µM Y-27632, and 10 ng/mL KGF or EGF for 56–84 days. TP63-positive cells were collected by FACS. RNA extraction was performed per in chip inside 10x Chromium controller. cDNA libraries were prepared as 118 bp paired-end reads using the Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics)