Pluripotent Stem Cell Differentiation Assay

The pluripotent stem cell lines, hESCs (WA09) and hiPSCs (MR90-1) from WiCell Research Institute, WI, USA were used at passages 29 to 33. Cells were cultured in mTeSR1 (STEMCELL Technologies Canada Inc., Vancouver, BC) in Matrigel Matrix (Corning, NY, US) as recommended by the manufacturer. The hESCs and hiPSCs were either treated with 10 μM of Y-27632 ROCK inhibitor or not. Samples were collected at different time-points, 0-untreated, 12, 24, 48, 96 hours (h) and tested over the expression of pluripotency markers (Fig. 1A). The earliest time point that meaningful analysis could be performed is 12 h as the single cells need sufficient time to attach to the culture surface. Samples treated with ROCK inhibitor (Y-27632) where cultured in mTeSR1 with 10 μΜ Y-27632 (dihydrochloride - Tocris Bioscience, Bristol, UK) for 2 hours. Thereafter, cells were detached with the use of Accutase (StemPro - Thermo Fisher Scientific, MA, USA), colonies were dissociated into single cells by pipetting22 (link)23 (link)24 (link), seeded at 30–40 * 10⁴ cells/cm² density on Matrigel and cultured in mTeSR1 with 10 μΜ Y-27632 up to 96 h. Samples from three independent experiments (N = 3) were collected.