Y 27632

All animal experiments were approved by the Ethics Committee of Animal Experimentation of Kyoto Prefectural University of Medicine (permission number M26-237). All injections with needles were performed under anesthesia, and all efforts were made to minimize suffering. Pax3^GFP/+, MyoD-CreIZ, and Rosa26^{CAG-LSL-tdTomato/+} mice were used for obtaining skeletal muscle cells, and female Dmd^−/y crossed with male NSG mice (Sato et al., 2014 (link), Charles River) were used for the transplanted donors. Male Dmd^−/y;NSG mice were used for all experiments at the indicated ages.
Eight-week-old Dmd^−/y;NSG host male mice were used for engraftment of freshly isolated or cultured cells derived from hiPSCs (5.0 × 10⁵ cells per 20 μL of PBS with 2% horse serum [Sigma]), and 10 μM Y-27632 (Nacalai Tesque) into TA muscle fibers. Mice were anesthetized with diethyl ether prior to engraftment. TA muscle was removed 4 weeks after transplantation, fixed, and stained as described below. Three independent mice for the transplantation and ten sections of each transplanted mouse were analyzed.

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.

Tet-mCherry or Tet-MyoD hiPSCs were cultured on Easy iMatrix-511 silk-coated plates (#892024, Nippi) in StemFit medium (AK02N, Ajinomoto) containing 100 μg/mL G418 (#938044, NacalaiTesque) or 0.5 μg/mL puromycin dihydrochloride (160-23151, Wako Chemicals). Cells were passaged every 7 days using Accutase (#12679-54, NacalaiTesque) and seeded on Easy iMatrix-511 silk-coated 6-well plates in the presence of 10 μM Y-27632 (NacalaiTesque) at a density of 1.5×104 cells/well for the first 2 days after plating. At 48 h after passaging, Y-27632 was removed and replaced with StemFit medium containing the appropriate antibiotic.

Organoid-formation assays were performed as previously described^{34 (link)} with some modifications. Sort-purified crypt epithelial cells were mixed into Matrigel (3000–5000 cells/10 µl) containing 1 µM Jagged1 peptide, and then 10 µl of the sorted cell/Matrigel mixture was seeded into each well of a 96-well plate. The Matrigel was allowed to solidify for 15 min in a 37 °C incubator and then was overlaid with 100 µl culture medium containing advanced DMEM/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, Glutamax, 1× N2, 1× B27 (all from Invitrogen), 1 µM N-acetylcysteine (Sigma), 50 ng/ml EGF, 100 ng/ml Noggin (Peprotech), 10% RspoI-conditioned medium (culture supernatant of the 293T-HA-RspoI-Fc cell line, provided by Calvin Kuo of Stanford University), 500 ng/ml Epiregulin, 10 µM Y-27632 (first 2 days, Nacalai Tesque), and 3 µM CHIR-99021 (Axon Medchem) (hereafter referred to as NER + CH + Ereg Medium), and the cells were cultured for 7 days. The medium was changed every other day. Microscopic images of the organoid cultures were obtained with a Keyence BZ-X700.

Crypt isolation from the small intestinal samples (jejunum) and crypt embedding in Matrigel (CORNING, Corning, NY, USA) were performed as previously described [12 (link)]. After Matrigel polymerization, crypts were covered with medium of the IF condition. The medium was supplemented with 10 mM Y-27632 (Nacalai tesque, Kyoto, Japan) for the first three days of culture. The medium was replaced with fresh medium every two to three days. The organoids were passaged every six to nine days using TrypLE Express (Thermo Fisher Scientific) for dissociation into a single cell. In the differentiation culture of organoids, the organoids were cultured in the DI condition or IL-4 or IL-13 condition for 72 h after growth under the IF condition for six to nine days.

The hESC lines, H1 and H9, and hiPSC lines, 253G4 and 4A (HiPS-RIKEN-4A), were maintained in the human ESC medium as previously described.^{23 (link)} The hESC lines (H1 and H9) were purchased from WiCell Research Institute (WI, United States). The cell lines 253G4 and 4A were kindly provided by Prof. Shinya Yamanaka (Kyoto University, Kyoto, Japan) and Dr. Yukio Nakamura (RIKEN BioResource Center, Tsukuba, Japan), respectively. All hPSC lines were transferred to feeder-free conditions. The hPSC colonies were detached from the feeder cells by CTK solution (0.1% collagenase IV, 0.25% trypsin, 20% KSR, and 1 mM CaCl₂ in phosphate-buffered saline [PBS]). They were also passed through a 40-μm cell strainer to remove feeder cells, and the colonies remaining on the cell strainer were plated on vitronectin-coated (VTN; Life Technologies), Matrigel-coated (BD Matrigel hESC-qualified Matrix; BD Biosciences) or iMatrix-511-coated (nippi, Inc.) dishes in StemMACS™ iPS-Brew XF (Miltenyi Biotec) or Pluripro MATRIX-coated (CELL guidance systems) dishes in Pluripro media (CELL guidance systems). For single-cell-state culture, hPSC colonies were detached using Accutase (Innovative Cell Technologies) and were plated on VTN-, Matrigel-, or iMatrix-511-coated dishes in StemMACS iPS-Brew XF or Pluripro MATRIX-coated dishes in Pluripro media with 10 μM Y-27632 (Nacalai Tesque).

hiPSCs (414C2 line) were cultured for 12 days in adhesion cultures with mouse embryonic fibroblasts and then allowed to form embryonic bodies in floating culture for 30 days. Aggregated cells were differentiated into NS/PCs derived from hiPSC-NS/PCs using various factors during each day of the incubation period (Okada et al., 2008 (link)).
hiPSCs (201B7 line) were pretreated for 6 days with 3 μM SB431542 (Tocris, 301836-41-9) and 150 nM LDN193189 (StemRD, 1062368-24-4). The cells were then dissociated and seeded at a density of 1×10⁵ cells per milliliter in ultra-low-attachment culture dishes (Corning) in neuronal induction medium consisting of medium hormone mix (MHM) (Okada et al., 2008 (link)) supplemented with 2% B27 supplement without vitamin A (Thermo Fisher, 17504-044), 20 ng/mL FGF-2, 10 μM Y27632 (Nacalai Tesque, 08945-71), 1 μM retinoic acid (RA; Sigma, R2625-1G), 3 μM CHIR 99021 (Reprocell, 04-0004) and 10 μM SB431542 (Calbiochem, 301836-41-9) in a hypoxic and humidified atmosphere (4% O₂, 5% CO₂) for 6 days. The formed neurospheres were passaged by dissociation into single cells and then cultured in slightly modified neuronal induction medium, MHM supplemented with 2% B27 without vitamin A, 20 ng/mL FGF-2, 10 μM Y27632, and 1 μM RA for 6 days under 4% O₂ (hypoxic) conditions.