Extended Data Figure 1: The effects of culture parameters on epiblast explants.

Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a, In EpiSC derivation medium containing 20% KnockOut serum replacement (KSR), 20 ng ml⁻¹ Activin-A and 12 ng ml⁻¹ FGF2. Day 3 outgrowth was stained with pluripotency markers OCT4 and SSEA-1. b, In N2B27 media supplemented with indicated growth factors and small molecules. After 4 days, outgrowths of plated epiblasts were stained for OCT4 and SSEA-1. c, Percentages of SSEA-1⁺/OCT4⁺ cells in day 4 epiblast outgrowths in N2B27^KSR+F/A and N2B27^F/R1 culture conditions. A simple randomization method was applied to randomly pick the microscope fields of views for counting the number of SSEA-1⁺/OCT4⁺ cells and total cell numbers. PC, phase contrast. For examining different culture parameter effects, all isolated E5.75 epiblasts were pooled together and randomly allocated to each condition.