Mtesr plus serum free medium

The iPSK3 cell line was obtained by transfecting human foreskin fibroblasts with plasmid DNA encoding reprogramming factors octamer-binding transcription factor 4 (OCT4), NANOG, SRY-box transcription factor 2 (SOX2), and LIN28 (kindly provided by Dr. Stephen Duncan, Medical College of Wisconsin) [53 (link),54 (link)]. The cells were cultured in mTeSR Plus serum-free medium (StemCell Technologies, Inc., Vancouver, BC, Canada) on a growth-factor-reduced Geltrex-coated surface (Life Technologies, Carlsbad, CA, USA) [41 (link),55 (link)]. Before seeding, 1 mL of 1% Geltrex was added to a 6-well attachment plate. Coating was done at 37 °C for at least one hour. The coating solution was then removed, and 1.5 × 10⁶ cells were seeded in 3 mL of fresh mTeSR^TM Plus medium with 10 µM ROCK inhibitor Y-27632 (dilution, 1:1000). After 24 h, Y-27632 was removed and the media were replaced with fresh mTeSR^TM Plus media. The media were changed daily for 4–5 days until the cells were ready to be passaged again.

Human foreskin fibroblasts were transfected with plasmid DNA encoding reprogramming factors octamer-binding transcription factor 4 (OCT4), NANOG, SRY-box transcription factor 2 (SOX2), and LIN28 to produce human iPSK3 cells (kindly provided by Dr. Stephen Duncan, Medical College of Wisconsin)^{54 (link),55 (link)}. The hiPSCs were maintained in mTeSR Plus serum free medium (StemCell Technologies, Inc., Vancouver, Canada) on growth factor-reduced Geltrex-coated surface (Life Technologies). The cells were passaged every seven days using Accutase and seeded at 1 × 10⁶ cells per well of six-well plate in the presence of rho-associated protein kinase (ROCK) inhibitor Y27632 (10 μM, Sigma) for the first 24 h^{56 (link),57 (link)}.