Research Article

In vitro culture of cynomolgus monkey embryos beyond early gastrulation

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Science  15 Nov 2019:
Vol. 366, Issue 6467, eaax7890
DOI: 10.1126/science.aax7890

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In vitro development of monkey embryos

Owing to technical and ethical limitations, the molecular and cellular mechanisms underlying primate gastrulation are far from clear (see the Perspective by Tam). Two independent studies used an in vitro culture system to study cynomolgus monkey embryo postimplantation development up to and beyond gastrulation (day 9 to day 20). Niu et al. observed in vivo morphogenetic events and used single-cell RNA sequencing and single-cell chromatin accessibility to study the distinct cell lineages in developing embryos. Ma et al. also observed that key events of in vivo early development were recapitulated in their system, and single-cell RNA-sequencing analysis revealed molecular signatures of postimplantation cell types. These systems will help elucidate the dynamics and regulation of gastrulation in primates, including possible relevance to human development.

Science, this issue p. eaaw5754, p. eaax7890; see also p. 798

Structured Abstract


Gastrulation is a landmark event in development that involves a complex series of molecular, physical, and energetic remodeling transitions in early embryogenesis. Processes vary among species, leading to the diversity of animal forms on Earth. A dearth of primate embryo samples at the gastrulation stage has limited our understanding of this critical event in primates. Recently, human embryos were grown in culture for 12 to 13 days. Many governments and international organizations have recommended that human embryos should not be allowed to grow beyond 14 days in vitro. Therefore, it is expected that analysis of nonhuman primate embryo model systems will elucidate mechanisms underlying gastrulation and hopefully shed light on human development and the processes associated with defects and disease that arise during early development.


Monkeys have long been considered a reliable animal model with which to study human physiological and pathological events because of their high degree of similarity to humans in both genomic and morphological characteristics. Therefore, we developed a system that supports the growth of cynomolgus monkey embryos in vitro for up to 20 days postfertilization (d.p.f.). Histological and immunofluorescent staining, combined with single-cell RNA-sequencing (RNA-seq) analysis, demonstrated that these in vitro–cultured (IVC) monkey embryos developed beyond early gastrulation and recapitulated key events of early primate in vivo postimplantation development.


We cultured cynomolgus monkey blastocysts with a mature blastocoel (d.p.f. 7 to 8) using the IVC system. At d.p.f. 13 to 14, a bilaminar disc–like structure appeared in ~27.7 ± 3.2% of the IVC embryos (n = 167, 26 experiments). At d.p.f. 15 to 16, the disc-like structure was clearly observable under the optical microscope. Some of the embryos successfully developed to d.p.f. 20 in vitro. Hematoxylin and eosin (H&E) and immunofluorescent staining confirmed that the IVC embryos recapitulated the major hallmarks of in vivo early postimplantation development: segregation of the epiblast (OCT4+, NANOG+) and hypoblast (GATA6+) lineages; formation of the amniotic and yolk sac cavities; appearance of the presumptive primordial germ cells (SOX17+, TFAP2C+, BLIMP1+); establishment of the anterior-posterior axis (asymmetric localization of OTX2+ cells); and gastrulation itself (gastrulating cells: T+/OCT4+, VIMENTIN+/T+/OCT4+). Furthermore, single-cell RNA-seq analysis showed that the monkey IVC embryos were similar to their in vivo counterparts in gene expression profiles and cell types, including presumptive parietal trophoblasts, extraembryonic mesenchyme cells, postimplantation early and late epiblasts (E-EPI and L-EPI, respectively), visceral and yolk sac endoderm, early primordial germ cells (E-PGC), early gastrulating cells (E-Gast), late gastrulating cells 1 and 2 (L-Gast1 and L-Gast2, respectively), early amnion cells (E-AM), and late amnion cells 1 and 2 (L-AM1 and L-AM2, respectively).


We have established an IVC system that can support the development of cynomolgus monkey embryos beyond early gastrulation in vitro. The IVC embryos recapitulate numerous key events of in vivo early postimplantation development of primate embryos. Single-cell RNA-seq analysis reveals the molecular signatures of several cell types of primate early postimplantation embryos, including amnion cells. The monkey IVC system provides a platform for future studies of molecular signatures and mechanisms of early embryogenesis that are specific to primates with potential relevance to human diseases that arise during early development.

Monkey embryos grow in vitro beyond early gastrulation.

IVC embryos were stained with antibodies for OCT4 (green) and GATA6 (red) (d.p.f. 13 to 14 or 19) or H&E (d.p.f. 20). Single-cell transcriptome analysis revealed the similarities among cell types in the in vitro and in vivo monkey early embryos and the developmental trajectory of epiblast derivatives. Scale bars, 100 μm.


Gastrulation is a key event in embryonic development when the germ layers are specified and the basic animal body plan is established. The complexities of primate gastrulation remain a mystery because of the difficulties in accessing primate embryos at this stage. Here, we report the establishment of an in vitro culture (IVC) system that supports the continuous development of cynomolgus monkey blastocysts beyond early gastrulation up to 20 days after fertilization. The IVC embryos highly recapitulated the key events of in vivo early postimplantation development, including segregation of the epiblast and hypoblast, formation of the amniotic and yolk sac cavities, appearance of the primordial germ cells, and establishment of the anterior-posterior axis. Single-cell RNA-sequencing analyses of the IVC embryos provide information about lineage specification during primate early postimplantation development. This system provides a platform with which to explore the characteristics and mechanisms of early postimplantation embryogenesis in primates with possible conservation of cell movements and lineages in human embryogenesis.

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