Culturing Primed Human Pluripotent Stem Cells

HEK293T cells and ICR mouse embryonic fibroblast-derived feeders were maintained in DMEM (Corning) supplemented with 10% fetal bovine serum (NATOCOR). Primed human PSCs, including H9 ESCs and UH10 iPSCs^{50 (link),51 (link)} were routinely cultured in mTeSR^TM1 medium (STEMCELL). For passaging, primed PSCs were washed with DPBS (Hyclone) once and treated with 0.5 mM EDTA (Invitrogen, 15575020) for 5 minutes. Then, EDTA was removed and cells were passaged as small clumps using a Pasteur pipette. Human TSC^BT were cultured in TSC medium^{15 (link)} (DMEM/F12 supplemented with N2 and B27, penicillin-streptomycin, Glutamax, β-mercaptoethanol, 1.5 µg/ml L-ascorbic acid, 50 ng/ml EGF (PeproTech), 0.5 µM A83-01 (Selleck), 1 µM SB431542 (Selleck), 2 µM CHIR99021 (Axon), 0.8 mM VPA (Vetec) and 5 µM Y-27632 (Axon) supplemented with 10 µM Y-27632. Human H9 ESCs were purchased from WiCell Research Institute, human UH10 iPSCs were provided by Dr. Guangjin Pan (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China), and TSC^BT were provided by Dr. Hiroaki Okae and Dr. Takahiro Arima (Department of Informative Genetics, Tohoku University Graduate School of Medicine). All cell lines were negative for mycoplasma.