Extended Data Figure 8. 3D in vitro modelling of the marmoset EmDisc.

a, Schematic overview of interphase culture. To model self-organisation of conventional cmPSCs (common marmoset pluripotent stem cells) in 3D culture, cmPSCs were seeded on a 100% Matrigel base overlaid with 1% Matrigel dissolved in N2B27 media supplemented with signalling factors. Interphase culture was amenable to probing signalling requirements that promote anterior embryonic disc-like pluripotency or differentiation into the germ layers of the gastrulating embryo. All experiments were performed in two different cell lines (cell line #1 (New4f), N=2 and cell line #2 (New2f), N=2) showing consistent results.

b, Heatmap of marker genes used for molecular characterisation of interphase culture structures. Relative mRNA levels were centred and scaled across all marmoset in vivo samples.

c, Summary schematic of NODAL and FGF signalling in the marmoset embryo. The visceral endoderm is the primary source of NODAL and IGF1 in the marmoset embryo, while the EmDisc expresses low levels of FGF4. Relative mRNA expression gradients summarised in CS6 cross section.

d, Time series brightfield images of interphase culture with FGF (100ng/ml) and Activin A (20ng/ml). FGF/Activin A culture provides a signalling environment that mimics high NODAL and IGF1 from the VE. Structures formed columnar epithelial cysts, reminiscent of the embryonic disc. Structures first open a lumen at day 3 and expand up to day 6.

e-f, Molecular characterisation of EmDisc-like structures at day 4. Representative maximum projection images from immunostaining at day 4 for pluripotency (SOX2), early gastrulation (TBXT), amnion (TFAP2C, TFAP2A), endoderm (SOX17) or mesoderm (CDX2) markers. EmDisc-like structures homogenously expressed SOX2, with heterogeneous low expression of TBXT, SOX17 and TFAP2C indicative of priming toward gastrulation and rare emergence of endoderm. Pluripotent EmDisc-like structures support a role for FGF and Activin/NODAL signalling in promoting pluripotency in the EmDisc.

g, Summary schematic of canonical WNT signalling in the marmoset embryo. The posterior EmDisc, stalk and PGCs express WNT3. mRNA expression gradients summarised in CS6 cross section (left). Time series brightfield images of interphase culture with FGF and Activin A + CHIR (CHIR99021, WNT agonist) (right). The emergence of differentiated populations was evident at day 4.

h-i, Molecular characterisation of WNT-treated EmDisc-like structures at day 4. Representative maximum projection images from immunostaining at day 4 from staining for pluripotency (SOX2), early gastrulation (TBXT), amnion (TFAP2C, TFAP2A), endoderm (SOX17) or mesoderm (CDX2) markers. Structures exhibited loss of SOX2 expression and upregulation of TBXT and SOX17 in comparison to FGF/Activin A culture, consistent with differentiation into amnion, endoderm, and mesoderm populations.

j, Summary schematic of WNT inhibition in the marmoset embryo. The VE and Amnion express canonical WNT inhibitor DKK1. mRNA expression gradients summarised in CS6 cross section (left). Time series brightfield images of interphase culture with FGF and Activin A + IWP-2 (right). Similar to FGF/Activin A culture, structures first open a lumen at day 3 and expand up to day 6.

k-l, Molecular characterisation of WNT-inhibited EmDisc-like structures at day 4. Representative maximum projection images from immunostaining at day 4 from staining for pluripotency (SOX2), early gastrulation (TBXT), amnion (TFAP2C, TFAP2A), endoderm (SOX17) or mesoderm (CDX2) markers. EmDisc-like structures homogenously expressed SOX2 and downregulated TBXT and SOX17 in comparison to FGF/Activin A culture, consistent with a role for WNT inhibition in preserving pluripotency in the EmDisc.

m, Summary schematic of BMP signalling in the marmoset embryo.The ExMes, amnion, and PGCs are sources of BMP4 in the embryo. mRNA expression gradients summarised in CS6 cross section (left). Time series brightfield images of interphase culture with FGF and Activin A + BMP4 (right).The emergence of disorganized, differentiated populations was evident at day 4.

n-o, Molecular characterisation of BMP-treated EmDisc-like structures at day 4.Representative maximum projection images from immunostaining at day 4 from staining for pluripotency (SOX2), early gastrulation (TBXT), amnion (TFAP2C, TFAP2A), endoderm (SOX17) or mesoderm (CDX2) markers. Structures exhibited loss of SOX2 expression and upregulation of TFAP2C, TFAP2A, CDX2 and SOX17 in comparison to FGF/Activin A culture, consistent with a mixed amnion and posteriorized primitive streak-like fate.

p, Summary schematic of BMP inhibition in the marmoset embryo. The VE expresses BMP inhibitor NOGGIN in the embryo. mRNA expression gradients summarised in CS6 cross section (left). Time series brightfield images of interphase culture with FGF and Activin A + BMP4 (right).Similar to FGF/Activin A culture, structures first open a lumen at day 3 and form homogenous spheroids.

q-r, Molecular characterisation of BMP-inhibited EmDisc-like structures at day 4.Representative maximum projection images from immunostaining at day 4 from staining for pluripotency (SOX2), early gastrulation (TBXT), amnion (TFAP2C, TFAP2A), endoderm (SOX17) or mesoderm (CDX2) markers. Scale bars represent 100 μm. EmDisc-like structures homogenously expressed SOX2 and downregulated TBXT, SOX17 and TFAP2C in comparison to FGF/Activin A culture. This is consistent with a role for BMP inhibition in preserving pluripotency in the EmDisc and inhibiting amnion differentiation.