Pd0325901

Three primed hESC lines were subjected to naive conversion on MEFs in low oxygen conditions in either: (i) naive human stem cell medium (NHSM) composed of basal hESC media supplemented with AlbumaxI, N2 supplement (Invitrogen), PD0325901 (1 μM, Cayman) and CHIR99021 (3 μM, Axon Medchem; known together as 2i), leukaemia inhibitory factor (LIF, 1000U, Sigma), bFGF, (8 ng ml⁻¹), SP600125 (10 μM, Tocris), SB203580 (10 μM, Tocris) and TGFβ (1 ng ml⁻¹, Peprotech)10 (link); (ii) RT consisting of high glucose DMEM-F12 (Invitrogen) further supplemented with histone deacetylase inhibitors, sodium butyrate (0.1 mM) and SAHA (50 nM) for first three passages followed by 2i and bFGF (10 ng ml⁻¹)11 (link) or (iii) our novel medium naive conversion medium (NCM) containing hESC media with added 2i, LIF, forskolin (10 μM, Sigma), ascorbic acid (50 ng ml⁻¹, Sigma) and bFGF(12 ng ml⁻¹)8 (link). Upon the appearance of domed-shaped colonies, the culture was passaged using 0.05% trypsin and once the culture was well established, cells were split at a ratio of 1:4 to 1:8 every 3 days. Single-cell clonogenicity was determined from the number of single-cells plated (upon dissociating primed and naive hESCs using trypsin without ROCKi) and the subsequent number of colonies formed. Doubling time was calculated using the formula:

EOAI3402143 (referred to as G9) was synthesized and provided by Evotec (UK), (Abingdon Oxfordshire, UK). Other reagents used in this study were obtained from the following sources: hemagglutinin-tagged ubiquitin vinyl methyl sulfone (HA-UbVS; Boston Biochem); Vemurafenib (PLX4032; Chemie Tek); PD0325901 (Cayman Chemical); MI-219 (A kind gift of Dr. Shaomeng Wang, University of Michigan). All reagents were made up and stored frozen as 10 mM stock solutions.

Mouse embryonic stem cell line (kind gift from Ivan Bedzhov, MPI Münster) were maintained under 5% CO₂ and 37 °C conditions on 10% gelatine-coated (Sigma, G1393) T25 flasks in DMEM high glucose medium (Sigma D5671) containing 15% FBS (Sigma S0615), 100 U/ml Penicillin-Streptomycin, 2 mM L-Glutamine, 1 mM sodium pyruvate (Sigma, S8636), 0.1 mM NEAA (Sigma, M7145), 0.1 mM 2-mercaptoethanol (Sigma, M3148). Maintenance medium was freshly supplemented with 0.44 nM mLif (Amsbio, AMS-263), 0.4  $\mu$ M PD 0325901 and 3  $\mu$ M CHIR 99021 inhibitors (Cayman Chemicals). Seeding cell number was set to 4.2 × 10⁵ cells (for 2-day interval passaging) or 1.5 × 10⁵ (for 3 day interval passaging). The cells carry a fluorescent marker in the nucleus (H2B–GFP⁺) and express a red Ca²⁺ sensor (R-GECO 1.0). The cell line was detected positive for myoplasm after the experiments were performed.