2.2. Stem Cell Culture Conditions

Murine embryonic (D3) and induced pluripotent iPS-3, stem cells were cultured in high glucose (25 mM)) DMEM that was supplemented with; 15% (v/v) embryonic stem cell FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol and 10 ng/mL LIF. Both cell lines were provided by the Rancourt lab [62]. Stem cells were co-cultured on inactivated murine embryonic fibroblast cells, as described next.

Murine embryonic fibroblast (MEF) cells were cultured on 0.1% gelatin (Sigma) coated tissue culture plates. These cells were grown in high glucose (25 mM) DMEM medium with 10% (v/v) FBS, 0.1 mM non-essential amino acids, 50 units/mL penicillin and 50 mg/mL streptomycin. MEFs were used for experimental purposes and as a feeder layer for stem-like cells. MEFs were prepared as a feeder layer and when they reached 100% confluency the cells were treated with 10 µg/mL mitomycin C (Sigma) for 2 h at 37 C. The cells were then washed with PBS and incubated in fresh medium overnight. Inactivated MEFs were used as feeder cells the following day [63]. When stem cells were required for experiments they were removed from MEFs. Once the stem cells reach 70–80% confluency on the gelatin plates the cells were dissociated and counted using a haemocytometer and Trypan blue. The cells were then aliquoted in appropriate amounts for subsequent experiments. Stocks of these cell lines were maintained in liquid nitrogen for long term storage in a 90% FBS 10% DMSO solution.

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] 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].

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] 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.