Nature | Vol 577 | 23 January 2020 | 537Article
A developmental landscape of 3D-cultured
human pre-gastrulation embryos
Lifeng Xiang1,2,3,4,7, Yu Yin1,3,4,7, Yun Zheng1,4,5,7, Yanping Ma2,7, Yonggang Li2,7, Zhigang Zhao^1 ,
Junqiang Guo1,5, Zongyong Ai1,4, Yuyu Niu1,4, Kui Duan1,4, Jingjing He1,4, Shuchao Ren^1 , Dan Wu^1 ,
Yun Bai^2 , Zhouchun Shang^6 , Xi Dai^6 , Weizhi Ji1,4* & Tianqing Li1,4*Our understanding of how human embryos develop before gastrulation, including
spatial self-organization and cell type ontogeny, remains limited by available
two-dimensional technological platforms^1 ,^2 that do not recapitulate the in vivo
conditions^3 –^5. Here we report a three-dimensional (3D) blastocyst-culture system that
enables human blastocyst development up to the primitive streak anlage stage. These
3D embryos mimic developmental landmarks and 3D architectures in vivo, including
the embryonic disc, amnion, basement membrane, primary and primate unique
secondary yolk sac, formation of anterior–posterior polarity and primitive streak
anlage. Using single-cell transcriptome profiling, we delineate ontology and
regulatory networks that underlie the segregation of epiblast, primitive endoderm
and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique
and characteristic phenotypes. After implantation, specific pathways and
transcription factors trigger the differentiation of cytotrophoblasts, extravillous
cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to
pluripotency upon implantation, and the transcriptome of these cells is maintained
until the generation of the primitive streak anlage. These developmental processes
are driven by different pluripotency factors. Together, findings from our 3D-culture
approach help to determine the molecular and morphogenetic developmental
landscape that occurs during human embryogenesis.Technical limitations preclude the precise delineation of early human
embryogenesis, such as architecture formation and cell-type specifica-
tion. Recent in vitro implantation platforms using a two-dimensional
(2D) culture approach have revealed some developmental landmarks
of early human embryos in vivo^1 ,^2. However, these 2D culture embryos
are largely flattened, which creates an imperfect model of normal
3D embryonic development in vivo and limits classification using
equivalent Carnegie stages^3 –^5. Although pluripotent stem cells can
model some phenotypes of the human amnion sac, amniogenesis or
organizer^6 –^8 , we still cannot authentically mimic human embryonic
development, especially for embryo lineage ontogeny. These existing
methods exclude crosstalk among different cell types in embryos.
Here, we report a 3D-culture system that enables development of the
human blastocyst up to the primitive streak anlage (PSA) stage. Using
the 3D platform, we reveal a developmental landscape of human pre-
gastrulation embryos.
3D architectures in 3D-cultured embryos
We used donated human embryos from surplus embryos after clinical
in vitro fertilization. We tested whether the culture media used for
2D human embryo cultures—IVC1 and IVC2^1 ,^2 —were suitable for 3D
culture of blastocysts. These media sustained only 6.3% of embryos
until 14 days post-fertilization (d.p.f.) using morphological embryo
observations (Extended Data Fig. 1a–f ). We then added sodium lactate,
sodium pyruvate and ROCK inhibitor (Y27632)^9 to IVC1 and IVC2 media,
resulting in modified IVC1 (mIVC1) and mIVC2, respectively. Cultures in
mIVC1 and mIVC2 sustained 23.4% of human blastocysts until 14 d.p.f.
(Extended Data Fig. 1a–f ).
We then designed a series of 3D extracellular matrix with Matrigel
embedding to identify an ideal 3D blastocyst-culture system. We veri-
fied embryonic development using morphological observations and
staining for specific lineage markers—OCT4 for the inner cell mass (ICM)
and epiblast (EPI), GATA6 for primitive endoderm/hypoblast (PrE) and
CK7 for trophoblast (TrB) (Extended Data Fig. 1g–j). We found that 10%
Matrigel yielded the best outcome and enabled 23.5% of blastocysts to
develop to 14 d.p.f. with normal embryonic structures (Extended Data
Fig. 1g–m). We used 10% Matrigel in conjunction with mIVC1 and mIVC2
to culture blastocysts unless noted otherwise (Fig. 1a).
Morphological observations revealed that we could culture human
blastocysts up to 14 d.p.f. using our 3D platform (Fig. 1b). Nearly all
blastocysts at 5–6 d.p.f. were positive for GATA6, but negative for CK7,
whereas the ICM expressed OCT4, NANOG, KLF17 and PRDM14 (Fig. 1c, d,
Extended Data Fig. 9b). At 7 or 8 d.p.f., GATA6, CK7 and OCT4 showedhttps://doi.org/10.1038/s41586-019-1875-y
Received: 24 December 2018
Accepted: 5 December 2019
Published online: 12 December 2019
(^1) Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China. (^2) Department of
Reproductive Medicine, The First People’s Hospital of Yunnan Province, Kunming, China.^3 Faculty of Environmental Science and Engineering, Kunming University of Science and Technology,
Kunming, China.^4 Yunnan Provincial Academy of Science and Technology, Kunming, China.^5 Faculty of Information Engineering and Automation, Kunming University of Science and
Technology, Kunming, China.^6 BGI-Shenzhen, Shenzhen, China.^7 These authors contributed equally: Lifeng Xiang, Yu Yin, Yun Zheng, Yanping Ma, Yonggang Li. *e-mail: [email protected]; [email protected]