For short-term pre-exposure, hPSCs were exposed for 4 days to naïve conditions. Naïve cocktails used were: CDK8/19i cocktail [0.4 μM of CNIO-CDK8/19 inhibitor and 20 ng/mL hrLIF (Peprotech)], as reported [20 (link)], or the 2i-based cocktail PXGL [1 μM PD0325901 (Axon Medchem), 2 μM Gö6983 (Selleckchem), 2 μM XAV939 (Selleckchem) and 20 ng/mL hrLIF (Peprotech)] [8 (link)]. TSCs derivation was performed as previously reported by Kojima and colleagues [23 (link)]. Naïve and primed hPSCs were single-cell dissociated by TrypLE Express, and 0.5 × 106 cells were seeded in a 6-well plated pre-coated with 5 mg/mL Collagen IV (#354233, Corning) and cultured in TS medium [DMEM/F12 (D6421, Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 0.2% FBS (Gibco), 0.3% BSA (Sigma), 1% ITS-X (Gibco), 1.5 μg/mL L-ascorbic acid (Sigma), 50 ng/mL EGF (PeproTech), 2 μM CHIR99021 (Axon Medchem), 0.5 μM A83-01 (Tocris, Bristol, UK), 1 μM SB431542 (PeproTech), 0.8 mM VPA (Sigma) and 5 μM Y-27632 (Selleckchem)] [24 (link)]. Cells were passaged every 5–7 days in single cells using TrypLE Express on pre-coated Collagen IV plates.
Bernad R., Lynch C.J., Urdinguio R.G., Stephan-Otto Attolini C., Fraga M.F, & Serrano M. (2021). Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19. Cells, 10(4), 876.
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