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 forOCT4 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.