Rho associated kinase rock inhibitor y 27632

Human embryonic stem cell lines H9, hESKM-05 [48 (link)], and iPS12 [49 (link)] were used as control cell lines. The hiPSMEN1-MNA-6, edited clones, and control ESCs were maintained in feeder-free conditions (mTeSR™1 medium, Matrigel hESC-qualified matrix) with daily medium change. For passaging, cells were treated with Gentle Cell Dissociation Reagent (Gibco) and replated on Matrigel hESC-qualified matrix in mTeSR™1 medium containing 10 µM Rho-associated kinase (ROCK) inhibitor Y-27632 (STEMCELL Technologies). Cells were cryopreserved in a freezing medium (10% DMSO in FBS) at −150 °C. All cell lines tested negative for mycoplasma contamination.

Human embryonic stem cell lines H9 (Wicell, Madison, WI, USA), H1 (Wicell, WA01) and induced pluripotent human stem cells BJ-EOS clone 4YA (provided by the Ellis lab) [64 (link)] were cultured in mTeSR complete medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 50 units/mL penicillin and 50 mg/mL streptomycin. These cells were cultured on Matrigel (Corning, New York, NY, USA) treated 60 mm plates as per the manufacturers specifications [64 (link)].
Cells were passaged by washing the cultures with Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS, Stem Cell Technologies) twice followed by accutase (Stem Cell Technologies) treatment. Once the cells were in a single cell suspension fresh medium was used to inhibit accutase activity. Cells were then counted and aliquoted for subsequent experiments. Cell were maintained in medium containing 10 μM Rho-associated kinase (ROCK) inhibitor (Y-27632; Stem Cell Technologies) for 24 h after passaging and then returned to their regular medium.
Human foreskin fibroblast cells (HFF) [64 (link)] were cultured on gelatin coated plates in high glucose (25 mM) DMEM medium supplemented with; 15% (v/v) embryonic stem cell FBS, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acid and 0.1 mM 2-mercaptoethanol.