Stemfit ak02 medium

The cell line Ts21-ES-GATA1-WT, in which a human chromosome 21 was transferred into the human ESC line, KhES-1-derived subline, and Ts21-ES-GATA1s, in which the GATA1 mutation was introduced into the KhES-1-derived subline and then a human chromosome 21 was transferred into the GATA1s-ES, were previously established [33 (link)]. TAM-iPS-GATA1s, which was generated from the blasts of TAM patients with DS, and TAM-iPS-GATA1-WT, in which the GATA1 mutation of TAM-iPS-GATA1s was repaired, were established as described previously [36 (link)]. All PSCs were cultured on 0.25 μg/cm² Laminin511-E8 fragment iMatrix-511 silk (Nippi, Tokyo, Japan)-coated culture plates with StemFit AK02 medium (Ajinomoto, Tokyo, Japan). For passage, the cells were dissociated into single cells with 0.5×TrypLE Select (Thermo Fisher Scientific, Waltham, MA, USA) and plated at 265 cells/cm². 10 μM Rock inhibitor Y-27632 (Nacalai Tesque, Kyoto, Japan) was used at the time of the plating, and the medium was exchanged with fresh AK02 medium without Y-27632 the next day.

To generate GD1 patient‐derived iPSCs, skin fibroblasts of the patient (29 years, male, GM00372) were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research. The donor subject is a compound heterozygote for the following mutations: one allele carries a single‐base mutation (1226A>G) in the GBA gene, which results in the amino acid substitution of serine for asparagine (N370S); the second allele carries an insertion of a second guanine at cDNA nucleotide 84 (84GG). Three iPSC clones (clones 1, 2, 3) from the same donor were generated with StemRNA‐NM reprogramming kit (Stemgent) at ReproCELL, Japan. As the healthy control, 201B7 iPSCs were obtained from CiRA (Kyoto, Japan).²⁸ To rescue the enzymatic defect of GBA1, wild‐type GBA1 cDNA was cloned into the PiggyBac expression vector and the tet‐inducible iPSC line was generated as described previously.²⁹ Undifferentiated iPSCs were maintained in StemFit AK‐02 medium (Ajinomoto, Tokyo, Japan) on iMatrix‐511 (Nippi, Tokyo, Japan)‐coated plates.

Commercially available hiPSC lines were used in this study (Supplementary Table 1). HiPSC lines were obtained from RIKEN Cell Bank (201B7, 253G1, 409B2, HiPS-RIKEN-1A, HiPS-RIKEN-2A, and HiPS-RIKEN-12A), American Type Culture Collection (ATCC-DYR0110 hiPSC and ATCC-HYR01103 hiPSC), JCRB Cell Bank (Tic), and System Biosciences (human mc-iPS). HiPSCs were screened for mycoplasma contamination and hiPSCs used in this study were mycoplasma-free. Undifferentiated hiPSCs were maintained on an iMatrix-511 (Nippi) in StemFit AK02 medium (Ajinomoto). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air.