Y 27632

Guide RNA was designed on the basis of the PAM 5′‐NGG‐3′ for SpCas9, which was inserted into pCAGmCherry‐gRNA vector (Addgene 87110) using Gibson assembly (NEB) following the hCRISPR gRNA synthesis protocol (https://media.addgene.org/data/93/40/adf4a4fe-5e77-11e2-9c30-003048dd6500.pdf). Approximately 2 × 10⁶ iPSCs were treated with Accutase and then electroporated undergoing 1200 V by Neon transfection system (Invitrogen) mixing 2.5 μg gRNA vector, 2.5 μg Cas9 plasmid (Addgene 44719) and 5 μg ssODN (IGE). The transfected iPSCs were plated on Matrigel‐coated 35 mm dish and cultured in mTeSR medium supplemented with 10 μm Y‐27632 (Sigma) for one day. At 48 hours after electroporation, the positive single cells were harvested through FASC (BD Aria III) resulting from gRNA with mCherry reporter, Cas9 with GFP reporter, and replated on Matrigel‐coated 35 mm dish at the density of 5 × 10³ cells per dish with 10 μm Y‐27632 for one day. At day 10, we picked part of every clone as DNA template, which were amplified by Pfu PCR MasterMix (Tiangen) and HBB primers. We identified these clones through the products from polymerase chain reaction (PCR) used for Sanger sequencing.

To prepare single Lgr5 cells, crypts from Lgr5e-creERT2 mouse were isolated by incubating the opened lengthwise small-intestine pieces in the Gentle Cell Dissociation reagent (STEMCELL Technologies) on a rocking platform, at 20 rpm for 15 min at RT. Next the tissue pieces were resuspend in 10 ml ice-cold PBS containing 0.1% BSA and pipette up and down to strip off the crypts. The suspension was filtered through a 70-μm cell strainer. Filtered crypts were resuspended in 5 ml of TryPLE Express (Gibco) supplemented with Y27632 (10 μM) (Sigma-Aldrich). Crypts were transferred into a C-tube and dissociated by pre-programmed GentleMACS (Miltenyi Biotec). Cell suspension of the dissociated crypts were centrifuged and resuspended in 1 ml ice-cold DMEM/F-12 with HEPES (15 mM) (Gibco/Life Technologies) supplemented with N-acetylcysteine (0.5 mM) (Sigma-Aldrich), Y27632 (10 μM) and 1% (wt/vol) BSA. The cell number was counted by a cell counter to obtain a concentration of 1–5 × 10⁶ cells/ml. Cells were stained with 7-AAD (BD Biosciences) and Annexin V (BioLegend). Lgr5 cells were sorted through a 100-μm nozzle using BD FACS Aria III (BD Bioscience) into DMEM/F-12 with HEPES (15 mM), supplemented with N-acetylcysteine (0.5 mM) and Y27632 (10 μM).

Purified human monoclonal recombinant AQP4-IgG rAb-53 (AQP4-IgG) was provided by Dr. Jeffrey Bennett (Univ. Colorado Denver) as described [45 (link)]. Control human IgG (control-IgG) was purchased from Pierce Biotechnology (Rockford, IL, USA). Human complement (HC) was purchased from Innovative Research (Novil, MI, USA). Test drugs included clobetasol, miconazole, benztropine, clemastine, fumarate, retinoic acid and citicolone (Sigma-Aldrich, St. Louis, MO, USA), enprofylline, olesoxime and quetiapine fumarate (Santa Cruz Biotechnology, Dallas, TX, USA), GC-1 and quercetin (Tocris Bioscience, Bristol, UK), fasudil (Tszchem, Lexington, MA, USA), and Y-27632 (BD Biosciences, San Jose, CA, USA); Triiodothyronine (T3, Calbiochem, Billerica, MA, USA) was used as positive control. Drugs were dissolved in 2.5 % DMSO + 2 % solutole in PBS. Unless otherwise specified all other chemicals and media were purchased from Sigma-Aldrich.

Control hiPSCs were provided by the Stanford Cardiovascular Institute (Stanford University Cardiovascular Institutes Biobank, Stanford, CA, USA; Hypertrophic Cardiomyopathy/MYH7/Control iPSC line) (4 (link)). All hiPSCs were maintained in StemFlex media (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cryopreserved hiPSCs were thawed and added to StemFlex media supplemented with 5 μM Y-27632 (BD Biosciences). hiPSCs were then plated onto 8.7 μg/cm² Matrigel-coated (GFR, BD Biosciences) 6-well dishes. hiPSCs were subsequently cultured at 37°C at 5% CO₂ until they were 70%–90% confluent with daily media changes before passaging. For passaging, hiPSCs were dissociated using Versene (Thermo Fisher Scientific), resuspended in StemFlex media, and replated onto Matrigel-coated plates (Corning).

hESC H1 line was cultured feeder-free on hESC-qualified Matrigel (BD Biosciences) in E8 media (Stemcell Technologies) with 30% of irradiated mouse embryonic fibroblasts (iMEFs) conditional media. Cells were passaged every 3–5 days at 80% confluent with TrypLE Express (Invitrogen). After dissociation, the cells were plated in E8 media with 10 μM Y-27632, (StemGent) for 24 h. After 24 h media without Y-27632 was replenished daily. iMEF conditional media was prepared by incubating iMEFs with hESC media without bFGF for 24 h for 7 days. Collected media were filtered, flash frozen and stored at −80 °C. To initiate differentiation, the cells were dissociated using TrypLE Express to single cells and seeded at 150,000 cell/cm2 onto 1:30 dilution of growth factor reduced Matrigel (BD Biosciences) in DMEM/F12 in E8-MEF conditional media with 10 µM Y-27632. Two days following seeding the differentiation was started. Day 1 cells were exposed to RPMI + 3 µM CHIR-99021 (Stemgent) + 100 ng/ml rhActivinA (R&D Systems). Days 2–3: + 100 ng/ml rhActivinA + 0.2% FBS. Day 4–5: + 2% FBS + 50 ng/ml KGF (Peprotech). Days 6–9: DMEM/B27 + 50 ng/ml KGF + 2 μM RA (Sigma) + 0.25 μM SANT-1 (Sigma) + 100 ng/ml rhNoggin (R&D Systems). Days 10–14: DMEM/B27 + PdBU (1 µM) + Alk5i (1 µM) + 100 ng/ml rhNoggin (R&D Systems). PPs are defined as Day 9 and EPs as Day 14 of differentiation, respectively.