538 | Nature | Vol 577 | 23 January 2020
Article
mutually exclusive expression (Fig. 1e, f), which indicates that EPI,
PrE and TrB cells gained greater molecular and physical specificity.
TrB cells displayed strong filamentous CK7 staining (Fig. 1f). EPI cells
began to polarize and rearrange radially^10 (Fig. 1f). During development,
the human yolk sac has two developmental phases: a primary yolk sac
(PYS) develops between 7 and 9 d.p.f., followed by the formation of
the secondary yolk sac (SYS) at 12–13 d.p.f. (ref.^11 ). We did observe the
appearance of a small PYS surrounded by GATA6+ PrEs (Fig. 1f, Extended
Data Fig. 2a). At 10 d.p.f., radial arrangements indicated polarity and
epithelialization in EPIs by organized distributions of podocalyxin
(PODXL)^12 (Fig. 1g, h). We detected a distinct PYS (Fig. 1g, Extended Data
Fig. 2b). At 12 d.p.f., an amniotic cavity distinctly separated a group of
thinner squamous amniotic epithelium (AME) from more columnar EPIs
(Fig. 1i). By contrast, the amniotic cavity and yolk sac in 2D-cultured
embryos appeared to collapse at this stage^2. An obvious AME–EPI sepa-
ration was not observed in 2D-cultured embryos^1 ,^2 ,^6.
At 14 d.p.f., embryonic diameter and thickness measured over
500 μm and 400 μm, respectively (Extended Data Fig. 1k). The bilaminar
disc maintained continuous growth with an obvious SYS (Fig. 1j). As a
distinctive feature of anthropoid primates, the SYS is composed of
irregular visceral endoderm and squamous parietal endoderm based
on nuclear shape^13 (Fig. 1j, k), which shows considerable concordance
with anatomical descriptions from in vivo monkey and human 13–14-
d.p.f. embryos^11 ,^13. Squamous parietal endoderm expressed GATA6
(Fig. 1k), which suggests that it arises from PrEs that rapidly give rise
to the visceral endoderm and parietal endoderm after implantation^14.
Embryos displayed a 3D spherical structure with a disc-shaped bilami-
nar structure, SYS and amnion, and with the TrBs on the amnion side
of the embryo initiating trophoblast differentiation and undergoing
spatial and asymmetric development to concentrate at the amnion side
(Extended Data Fig. 1n–q, Supplementary Videos 1, 2). Together, our
3D platform can support self-organization with spatial architectures
of human embryos.Delineating lineage by transcriptome
We performed 557 single-cell RNA sequencing (scRNA-seq) from 42
embryos at 7 developmental stages to scan the transcriptome of our
3D cultured embryos (Fig. 2a). Following quality control and stringent
filtering, we used 555 single cells with 23,270 genes for subsequent
analyses (Extended Data Fig. 3a–c). t-distributed stochastic neighbour
embedding (t-SNE) analyses revealed seven clusters, identified as ICM,
EPI, PrE, TrB (including cytotrophoblasts (CTBs), syncytiotrophoblasts
(STBs) and extravillous cytotrophoblasts (EVTs)) and PSA-EPI based on
lineage-specific marker expression and developmental time (Fig. 2b–d).
Continuous transcriptome shifts from 6 to 14 d.p.f. reflected a tran-
sition from pre- to post-implantation (Fig. 2d). Integrated analysis
of scRNA-seq data from different embryo sources^15 –^18 revealed thatLow-attachment platek2k16 d.p.f.6 d.p.f. 7 d.p.f.8 d.p.f. 9 d.p.f. 12 d.p.f. 14 d.p.f.b
10% Matrigel 10% Matrigel 10% MatrigelmIVC1 mIVC2 mIVC2 mIVC28 d.p.f. 10 d.p.f.10 d.p.f.DAPI OCT4 CK7 GATA6 Merge12 d.p.f.AC ACAME
ACAMEPYS AMEPYS PYS
ACSYS SYS SYS
AC ACPYS14 d.p.f.NANOG PRDM14ACPYSc daeMaximum projectionOCT4 PODXL β-catening Maximum projection PODXLf h8 d.p.f. 9 d.p.f. 10 d.p.f. 11 d.p.f. 12 d.p.f. 13 d.p.f. 14 d.p.f.ijkGATA6 OCT4OCT4 CK7 GATA6VE k1k2OCT4SYSPE5–6 d.p.f. 5–6 d.p.f. 7 d.p.f.Fig. 1 | Human embryos self-organize in 3D architectures using our
3D blastocyst-culture conditions. a, Schematic of our in vitro 3D-culture
platform for human blastocysts. b, Bright-field images of human blastocysts
developing up to 14 d.p.f. c–e, Immunostaining of 5–6-d.p.f. (c, d, 3 out of 3
embryos) and 7-d.p.f. (e, 2 out of 2 embryos) blastocysts with markers as shown.
Red arrowheads (insets) denote ICM; white arrowheads denote
NANOG+PRDM14+ cells. f–h, Representative staining of an 8-d.p.f. (f, 3 out of 3
embryos) and a 10-d.p.f. (g, 4 out of 5 embryos; h, 3 out of 3 embryos) embryo
(Extended Data Fig. 2). The white and red arrows in f denote epiblast and
primary yolk sac, respectively. The white arrowhead in g denotes the amniotic
c av it y. i, Representative staining of a 12-d.p.f. embryo (6 out of 8 embryos). Red
arrowheads denote EPIs; white arrows denote AME. j, Representative staining
of 14-d.p.f. embryos (4 out of 6 embryos). k, Magnification of the square shown
in j, rotated 90° clockwise. AC, amniotic cavity; PE, parietal endoderm (white
arrows) (k2); VE, visceral endoderm (red arrows) (k1). Scale bars, 100 μm (b),
50 μm (c–g, i, j) or 10 μm (h). See also Extended Data Figs. 1, 2.