Stemfit ak02n medium

SEES-3 human ES cells were obtained from the National Center for Child Health and Development, Japan [18 (link)], and cultured under feeder-free conditions in StemFit AK02N medium (Ajinomoto, Tokyo, Japan) on laminin511 E8 fragment (iMatrix-511: Nippi, Tokyo, Japan)-coated dishes. All experiments were performed in accordance with the Guidelines for Derivation and Utilization of Human Embryonic Stem Cells by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Experimental protocols were approved by the Ethics Committee of Keio University School of Medicine for human stem cell experiments (No. 2012-01) and for recombinant DNA experiments (No. 24-010-12).

HiPSCs, namely 409B2 (RIKENBRC #HPS0076) were maintained in StemFit AK02N medium (Ajinomoto, Cat. No. RCAK02N), on a surface pre-coated with 0.5 mg/mL laminin-511 (Nippi, Cat. No. 892021) in PBS. For passage, cells were dissociated with Accumax (Innovative Cell Technologies, Cat. No. AM105-500), incubated 10 min at 37 °C, and routinely seeded at a density of 1 × 10³ cells/cm² in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor Y-27632 (Wako, Cat. No. 253-00513). After 48 h, cells were cultured without ROCK inhibitor. Cells were tested negative for mycoplasma contamination.

Peripheral blood cells from two patients with the MAPT R406W mutation were obtained (Ikeuchi et al., 2011 ), which were maintained in T cell medium, consisting of KBM502 medium (Kohjin Bio, Saitama, Japan) supplemented with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Waltham, MA, USA).
iPSCs were maintained on irradiated mouse embryonic fibroblasts or SNL 76/7 feeder cells in iPSC medium, consisting of DMEM/F12 medium (Wako, Osaka, Japan) supplemented with 0.08 mM MEM-Non Essential Amino Acid solution (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 20% (v/v) Knockout Serum Replacement (Thermo Fisher Scientific), 80 U/mL penicillin, 80 μg/μL streptomycin, 0.1 mM 2-mercaptoethanol, and 10 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA). Feeder-free iPSCs were maintained on culture dishes coated with 0.25–0.5 μg/μL iMatrix-511 (Laminin-511E8) (Wako) in StemFit AK02N medium (Ajinomoto, Tokyo, Japan). Medium was changed every day for iPSCs on feeder and every other day for feeder-free iPSCs.

The hiPSC cells, 454E2 line (HPS0077, RIKEN BRC, female)³² (link) and 1383D6 line (HPS1006, RIKEN BRC, male),³³ (link) were obtained from RIKEN BioResource Research Center (BRC), Cell Engineering Division (RIKEN cell bank). To culture hiPSCs under feeder-free conditions, StemFit AK02N medium (Ak02N, Ajinomoto, Japan) and polystyrene tissue culture plates coated with Laminin-511 fragment (iMatrix-511 silk; 892021, Matrixome, Japan) were used. On the first day after passage, AK02N medium supplemented with 10 μM Y-27632 (HY-10071; Wako, Japan) was used. The medium was changed every other day from the day after the passage. The iPSCs were passaged every 6–8 days using 0.5 × CTS TrypLE Select (A12859-01, Thermo Fisher Scientific, USA) supplemented with 0.5 mM EDTA in PBS(−) or only 0.5 mM EDTA in PBS(−).