Extended Data Figure 5: Functional analysis of Nanog-induced PGCLCs.

a, Experimental design (for b, c) for the derivation of EGC-like cells (EGCLCs). PGCLCs were induced with cytokines or by Nanog (+Dox) from male or female GOF-GFP EpiLCs carrying a constitutively active Kusabira-Orange reporter. On D4, aggregations were dissociated and cultured on MEF in PGC selection medium (LIF, SCF, bFGF, retinoic acid) for 5 days. After the selection, selected colonies were dissociated and transferred into ESC medium (2i LIF).

b, Experiment was performed as shown in (a). Left panel shows representative images of proliferating GFP+ve cells after 3 days of PGC selection. Right panel shows established EGCLCs after 3 passages in 2i LIF.

c, EGCLCs derived from D4 PGCLCs by Nanog expression were injected into blastocysts resulting in high contribution to chimeras at E9.5 as shown by Kusabira-Orange expression.

d, Experimental design (for e-g) for generating chimeras. PGCLCs were induced from a GOF-GFP ESC line expressing a fluorescent VENUS reporter constitutively and Nanog upon Dox addition (TVN2 cell line). On D4, aggregations were dissociated and SSEA1+ve and CD61+ve cells were sorted by FACS, injected into morulae and analysed on E9.5.

e, Representative brightfield, GFP/VENUS images of GOF-GFP or TVN2 cells during PGCLC induction by cytokines or Nanog (+Dox). Scale bars, 100μm.

f, FACS profile for SSEA1+ve and CD61+ve PGCLCs on D4 induced as described and shown in (d, e).

e, ESCs but not PGCLCs contribute efficiently to chimeras. ESCs or FACS-sorted Nanog-induced SSEA1+ve/CD61+ve PGCLCs were injected into morulae and representative brightfield/VENUS images from chimeras at E9.5 are shown.