Figure 2.

Naive ESC derived sEmbryos complete gastrulation and initiate neurulation and organogenesis stages within extraembryonic membranes

(A) Schematic depiction of the sEmbryo generation and culture protocol. DOX pre-induction (−1 day for Gata4 in 2i/Lif or AM, and −14 up to −1 day for Cdx2 in TSCm +/− LPA) and aggregation of 3 types of naive ESC-derived populations followed by culture in AM (with DOX in first 2 days), EUCM2, and EUCM for 8 days generates self-organized sEmbryos (iCdx2) that grow up to early organogenesis.

(B) Bright field images of sEmbryos at each day of the culture protocol compared to stage-matched natural embryos where indicated.

(C) On day 5, putative sEmbryos were transferred into an electronically controlled roller bottle ex utero culture platform set-up that was used for sEmbryo propagation until day 8.

(D) View of day 7 and day 8 sEmbryos cultured ex utero inside the roller culture bottles.

(E) Bright field images of day 8 sEmbryos growing ex utero within whole extraembryonic membranes (YS and Am).

(F) Day 8 sEmbryo (iCdx2) and E8.5 natural embryos after dissection and removal of extraembryonic membranes. Insets are enlargements of the dashed boxes.

(G) Image of empty YS obtained after continuous culture in IVC media (with KSR) and static culture-based protocol.

A, anterior; AC, amniotic cavity; Am, amnion; Al, allantois; AB, allantoic bud; BI, blood islands; EC, exocoelomic cavity; Epi, epiblast; EPC, ectoplacental cone; ExE, extraembryonic ectoderm; Fg, foregut pocket; H, heart; NFs, neural folds; NT, neural tube; OP, optic pit; P, posterior; PAC, pro-amniotic cavity; PS, primitive streak; S, somites; TB, tail bud; VE, visceral endoderm; YS, yolk sac.