Research Article

Dissecting primate early post-implantation development using long-term in vitro embryo culture

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Science  15 Nov 2019:
Vol. 366, Issue 6467, eaaw5754
DOI: 10.1126/science.aaw5754

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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


The period from peri-implantation to gastrulation is critical for mammalian embryogenesis. During this time, connections between embryonic and maternal tissues are set up, and the primary germ layers and body plan are established. There is a substantial gap in our knowledge of early human postimplantation development because of technological limitations and ethical considerations. To extend the study of human embryogenesis to the postimplantation period, an in vitro culture system has been established that extends human blastocyst development to the pregastrulation stage (up to12 days) after fertilization, and the molecular and cellular events are revealed.


With the general prohibition of growing human embryos beyond 14 days, closely related surrogate species can be examined. In addition, improvements are needed for primate embryo culture to support extended growth periods. The establishment of an in vitro culture system that enables the development of primate embryos beyond the implantation period provides an accessible way to study molecular and cellular mechanisms that underlie postimplantation development, including gastrulation.


In this study, we have modified a human embryo in vitro culture protocol that enables the development of cynomolgus monkey (long-tailed macaque) embryos to develop up to 20 days after fertilization. The cultured cynomolgus embryos recapitulated key primate in vivo morphogenetic events, including amniotic and yolk sac cavitation, embryonic and extraembryonic lineage specification, specification of primordial germ-like cells (PGCLCs), and primitive streak cells. We demonstrated that the amniotic lumenogenesis is accompanied by the polarization of the epiblast (EPI); however, the polarization of PE was not observed during yolk sac cavitation. We used single-cell RNA-sequencing to delineate the developmental trajectories of EPI, trophoblast, and primitive endoderm (PE) lineages. We observed that accompanying the transition from the naïve to primer state, the metabolic mode of oxidative phosphorylation is no longer used in the EPI cells. Furthermore, the trophoblast differentiates in a stepwise manner, and expression of the trophoectoderm marker CDX2 decreases rapidly after day 11 in the trophoblast but maintains in the amniotic epithelium cells. In addition, we identified two types of PE lineage. Coordinated interactions were observed among EPI, trophoblast, PE, and extraembryonic mesenchyme cells during the postimplantation period. Furthermore, we showed that PGCLCs specified in vitro are similar to early-stage PGCs in vivo. Using the single-cell assay for transposase-accessible chromatin followed by sequencing (scATAC-seq), we also identified EPI, trophoblast, PE, and EXMC lineages. Last, scATAC-seq revealed that distal regions of chromatin in the EPI lineage exhibited higher accessibility than in other cell types.


In this study, we show that monkey embryos show robust development beyond 14 days after fertilization, surviving until day 20 without support from maternal tissue. We also provide insights into the transcriptional programs and chromatin dynamics that underlie monkey post-implantation development. Our system provides a platform to analyze molecular and cellular dynamics during primate early development. Last, our data may help guide the development of improved differentiation protocols for primate pluripotent stem cells.

Monkey embryos cultured in vitro recapitulate primate postimplantation embryogenesis in vivo.

Scheme of monkey postimplantation embryogenesis cultured in vitro. ICM, inner cell mass; EPI, epiblast; PE, primitive endoderm; TE, trophectoderm; AMEC, amniotic epithelium cell; PGC, primordial germ cell; VE, visceral endoderm; YE, yolk-sac endoderm; EXMC, extra-embryonic mesenchyme cell; and GAS, gastrulating cell.


The transition from peri-implantation to gastrulation in mammals entails the specification and organization of the lineage progenitors into a body plan. Technical and ethical challenges have limited understanding of the cellular and molecular mechanisms that underlie this transition. We established a culture system that enabled the development of cynomolgus monkey embryos in vitro for up to 20 days. Cultured embryos underwent key primate developmental stages, including lineage segregation, bilaminar disc formation, amniotic and yolk sac cavitation, and primordial germ cell–like cell (PGCLC) differentiation. Single-cell RNA-sequencing analysis revealed development trajectories of primitive endoderm, trophectoderm, epiblast lineages, and PGCLCs. Analysis of single-cell chromatin accessibility identified transcription factors specifying each cell type. Our results reveal critical developmental events and complex molecular mechanisms underlying nonhuman primate embryogenesis in the early postimplantation period, with possible relevance to human development.

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