Fig. 1: Dual inhibition of Wnt and PKC enables derivation and long-term maintenance of chicken ES cells in combination with ovotransferrin.

a, Representative images of chicken blastodermal cells cultured in the indicated conditions for 3 days. n = 5 independent experiments. Scale bars, 50 μm. b, Representative images of chicken ES cells maintained in IWR-1 + Gö6983 + 3% yolk from unfertilized egg (Yolk/2i) at passage 35 (left panel, scale bar, 100 μm). The right panel provides a zoomed-in view of the dotted area from the left panel image (scale bar, 50 μm). n = 5 independent experiments. c, Schematic illustrating the methodology employed to identify the active component(s) within egg yolk that facilitates chicken ES cell self-renewal. The designation ‘30–80p’ refers to precipitates obtained at 30–80% saturation of ammonium sulfate. d, SDS–PAGE analysis of various fractions obtained from the ammonium sulfate precipitates. Whole and yolk plasma mixtures were within the 50 kDa to 100 kDa range. White dotted squares highlight the regions of the gels that were excised for mass-spectrometry analysis. n = 2 independent experiments. e, A representative image of RIR chicken ES cells maintained in OT/2i at passage 25 (left panel). The right panel provides a zoomed-in view of the dotted area from the image in the left panel. n = 10 independent experiments. Scale bars, 50 μm. MWCO, molecular-weight cutoff. Illustrations in c created using BioRender.com.