Tesr e8 medium

Human iPSC line 253G1 (Riken BRC Cell Bank, Tsukuba, Ibaraki, Japan) was established through retroviral transduction of OCT4, SOX2, and KLF4 into adult human dermal fibroblasts45 (link). The cells were cultured under feeder-free conditions using human embryonic stem cell (ESC)-qualified Matrigel (BD Biosciences, San Jose, CA, USA) and TeSR-E8 medium (Stemcell Technologies, Vancouver, BC, Canada) at 37 °C in an atmosphere containing 5% CO₂. For passage, iPSC colonies were dissociated into single cells using Accumax (Innovative Cell Technologies, San Diego, CA, USA) and cultured in TeSR-E8 medium supplemented with Y-27632 (ROCK inhibitor, 10 μM). The NPCs derived from iPSCs were cultured on poly-L-ornithine and Laminin (Thermo Fisher Scientific) coated dishes at 37 °C in an atmosphere containing 5% CO₂. The culture medium was Neural maintenance medium [NMM; a 1∶1 mixture of DMEM/F12 (Thermo Fisher Scientific) and Neurobasal (Thermo Fisher Scientific) containing N2 (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA; Thermo Fisher Scientific), 2-ME, PS]. For passage, NPCs were dissociated into single cells using Accumax and cultured in NMM supplemented with EGF (20 ng/ml), FGF2 (20 ng/ml) and Y-27632.

We used the human iPSC line 253G1 (Riken BRC Cell Bank, Tsukuba, Ibaraki, Japan), which was established through the retroviral transduction of Oct3/4, Sox2, and Klf4 into adult human dermal fibroblasts^{50 (link)}. The cell culture was performed as previously reported^{11 (link)}. Briefly, the cells were cultured under feeder-free conditions using human embryonic stem cell (ESC)-qualified Matrigel (BD Biosciences, San Jose, CA, USA) and TeSR-E8 medium (Stemcell Technologies, Vancouver, BC, Canada) at 37 °C in an atmosphere containing 5% CO₂. For passaging, the iPSC colonies were dissociated into single cells using Accumax (Innovative Cell Technologies, San Diego, CA, USA) and cultured in TeSR-E8 medium supplemented with the ROCK-inhibitor Y-27632 (10 μM) for the first two days.