Because mammalian embryos develop inside the uterus after implantation, they are practically inaccessible for direct observation and experimental analysis of the developmental process. To visualize and study the development of post-implantation embryos, it is necessary to develop a technology that maintains the viability and growth of embryos ex vivo in a controlled environment. This is especially the case for nonhuman primate embryos, which are likely to be adopted for modeling early human development. On pages 836 and 837 of this issue, Ma et al. (1) and Niu et al. (2), respectively, report in vitro culture methods for cynomolgus monkey embryos and demonstrate their utility for gaining insights into early primate embryo development.
The laboratory mouse is a road-tested animal model for mammalian development, and its use for investigating the development of pre- to early post-implantation embryos has been substantially enhanced by the ability to conduct experiments on embryos that are grown in culture for various durations between fertilization and early organogenesis (2–5). However, in view of the disparity of species-specific developmental features between mice and humans, there are reservations about the relevance of translating knowledge from mouse models of embryo development to primates (including humans). The use of human embryos for investigating early post-implantation development is limited by the ethical prerogative of the procurement of and experimentation on human embryos, as well as the technical barrier to sustaining normal growth and development beyond a few days. The ability to grow nonhuman primate embryos to post-implantation development provides a new model of early primate development. The similarity of genomic, anatomical, and physiological attributes between cynomolgus monkeys and humans posits this nonhuman primate embryo as an appropriate animal model for studying human development.
Pre-implantation cynomolgus embryos have been cultured in vitro from the fertilized oocyte, generated by in vitro fertilization (IVF), to the blastocyst at 7 days post-fertilization (dpf) (1, 2, 6). The availability of cultured IVF blastocysts removes the constraint of sourcing the scarce experimental material (blastocysts or post-implantation embryos) from pregnant animals for further culture and study. However, there has been little success in using present culture methods to support further development of the blastocyst.
The three-dimensional in vitro culture protocols reported by Ma et al. and Niu et al. have now extended the development of the blastocyst to the equivalent of 19 to 20 dpf (see the figure). By then, the cultured embryos display morphological signs of gastrulation: formation of the primitive streak (where cells move to the new germ layers), emergence of diverse cell types, and acquisition of anterior-posterior polarity. Gastrulation marks a critical developmental milestone of the mammalian embryo, occurring at ∼15 dpf in the cynomolgus embryo (6), when a diverse range of cell types of the embryonic and extraembryonic tissues are specified and are allocated to the primary germ layers.
Tracking the time course of embryo development in culture, as assessed by morphological landmarks and the appearance of constituent cell types, showed that these embryos have attained the morphogenetic milestones of their in vivo counterparts (1, 2). Overall, the embryos in culture recapitulated the in vivo development of the cynomolgus embryo up to the stage of early gastrulation. However, only a fraction (10 to 22%) of embryos developed normally past the initiation of gastrulation to 20 dpf. Experience in mouse embryo culture indicates that supporting development beyond gastrulation requires the provision of enriched culture media and unconstrained physical settings that can support extensive embryonic growth and the establishment of a functional fetal-maternal (placental) interface (5). Thus, there is a need to further optimize the present culture methods to enable extended post-gastrulation development.
Over ∼10 days of culture in vitro, the cynomolgus monkey blastocyst develops to a gastrulating embryo, which forms three germ layers and is surrounded by trophoblasts (which form the placenta) that allow it to adhere to the gel substrate to mimic implantation. Scale bar = 60 µm.
PHOTOS: MA ET AL. (1)
A wealth of knowledge about the delineation of embryonic and extraembryonic cell types, the specification of the germ line, and the transition of epiblast cells (which give rise to the germ cell layers) from pluripotency to lineage specification has been gleaned from single-cell transcriptome (gene expression) analysis of cynomolgus embryos in vivo (6, 7). The analysis of in vitro embryos by Niu et al. and Ma et al. demonstrates that the cells of the various embryonic and extraembryonic lineages found in the embryo in vivo were present in the cultured embryos. However, there is some discordance in the clustering of cell types according to transcriptomes between cultured and in vivo embryos, the spectrum of cell types may not be represented completely in vitro, and not all cells of the cultured embryos could be matched to known in vivo cell types. With the availability of multiple datasets (1, 2, 6, 7), it may be informative to reanalyze a merged dataset for collating the multitude of cell types in the in vitro embryo. This may reveal whether cells in an intermediate state in a known lineage, or cells of previously undescribed or transitory lineage, have been captured in the developing embryos in vitro.
A key outcome of gastrulation is the establishment of a blueprint of development according to a body plan that determines the composition and spatial assembly of progenitor cells (the building blocks of tissues and organs) and the form-shaping process of the body parts. The analysis of marker expression by Ma et al. and Niu et al. shows that the key cell types were detected at the appropriate developmental stage and location in the cynomolgus embryos in vitro. The regionalization of progenitor cells of different lineages or their descendants in the germ layers and extraembryonic tissues suggests that a body plan may have been established after gastrulation.
The in vitro model also offers the opportunity to perform an analysis of the spatiotemporal (by developmental stage) transcriptome of cell populations at defined positions in the cynomolgus monkey embryo. This may reveal the regionalization of molecular cell fates (8). One way to elucidate the cellular architecture of the body plan would be to map single cells to their inferred positions in the embryo by computational analysis of the transcriptome (9, 10) and integrate the results with data about the spatial distribution of cells of interest (11). Such a method could reveal the spatiotemporal genealogy that is embedded in the body plan. This information would add to the lineage trajectories of embryonic cells identified by Niu et al. and Ma et al. in cultured embryos. Analysis of the transcriptome and chromatin accessibility by Niu et al. has further uncovered the transcriptional, signaling, epigenomic, and molecular activity underpinning the specification and differentiation of cell lineages. Additional multiomics analysis of genomic function and molecular activity (12, 13) may generate a multidimensional body plan that could serve as the reference of cynomolgus development. These findings would engender testable hypotheses about the role of molecular drivers of lineage differentiation, tissue patterning, and morphogenesis.
Information generated from the in vitro cynomolgus embryo model will enhance understanding of the mechanisms and functional drivers of early embryogenesis and may outline a universal blueprint of primate development. Translating the knowledge of lineage specification and differentiation of the nonhuman primate embryo to stem cell research will guide efforts in directed differentiation of stem cells to produce clinically useful cell types and the generation of organoids for disease modeling and stem cell medicine. Thus, in vitro culture of cynomolgus post-implantation embryos is a valuable experimental tool for primate embryo research and stem cell biology.
The embryological findings of this model could provide information about errors of development in primate embryos. Blastocysts may be derived from in vitro fertilization of gametes that carry naturally occurring or engineered genetic changes or from genetically modified pre-implantation embryos. Modeling the phenotype and the causal mechanism of genetic diseases may inform how errors of development affect implantation, embryo viability, morphogenesis, and bodily function in ways that may lead to early pregnancy loss and birth defects. Such information could enable the development of preventive and therapeutic treatments and may improve the efficacy of assisted reproductive technologies.
The study of nonhuman primate embryos will offer a valuable cross-species reference of the developmental milestones of human embryos. It could also be informative about the “organismal potential” of synthetic embryos (i.e., whether they can develop like embryos in vivo) that are generated from stem cells—for example, embryoids from embryo-derived stem cells or induced pluripotent stem cells (14, 15). Although animal embryos grown for research may not raise the ethical concerns related to human embryo research and genome editing, it is imperative that the application of this primate embryo model adheres to the ethical principles of animal research and welfare.
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