Abstract
Human early embryonic development is the cornerstone of a healthy baby. Abnormal early embryonic development may lead to developmental and pregnancy-related disorders. Accordingly, understanding the developmental events and mechanisms of human early embryonic development is very important. However, attempts to reveal these events and mechanisms are greatly hindered by the extreme inaccessibility of in vivo early human embryos. Fortunately, the emergence of in vitro culture (IVC) systems for mammalian embryos provides an alternative strategy. In recent years, different two-dimensional and three-dimensional IVC systems have been developed for human embryos. Ethical limitations restrict the IVC of human embryos beyond 14 days, which makes non-human primate embryos an ideal model for studying primate developmental events. Different culture systems have supported the development of monkey embryos to days postfertilization 14 and 25, respectively. The successful recapitulation of in vivo developmental events by these IVC embryos has greatly enriched our understanding of human early embryonic development, which undoubtedly helps us to develop possible strategies to predict or treat various gestation-related diseases and birth defects. In this protocol, we establish different two-dimensional and three-dimensional IVC systems for primate embryos, provide step-by-step culture procedures and notes, and summarize the advantages and limitations of different culture systems. Replicating this protocol requires a moderate level of experience in mammalian embryo IVC, and the embryo culture requires strict adherence to the procedures we have described.
Key points
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This protocol describes the in vitro culture of cynomolgus monkey embryos from blastocyst to early organogenesis (up to 25 days postfertilization). Both two- and three-dimensional culture systems are described.
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Ethical limitations restrict human embryos beyond 14 days, which makes nonhuman primate embryos an ideal model.
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Data availability
Supporting data of this study can be found in our previous publications24 and are available from the corresponding author upon request.
References
Zhang, P. et al. Transcriptome profiling of human pre-implantation development. PLoS ONE 4, e7844 (2009).
Hertig, A. T., Rock, J. & Adams, E. C. A description of 34 human ova within the first 17 days of development. Am. J. Anat. 98, 435–493 (1956).
Sathananthan, H., Selvaraj, K. & Clark, J. The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro. Reprod. Biomed. Online 23, 227–233 (2011).
Müller, F. & O’Rahilly, R. The primitive streak, the caudal eminence and related structures in staged human embryos. Cells Tissues Organs 177, 2–20 (2004).
Rossant, J. & Tam, P. P. L. Early human embryonic development: blastocyst formation to gastrulation. Dev. Cell 57, 152–165 (2022).
Hill, M. A. Developing the digital Kyoto collection in education and research. Anat. Rec. 301, 998–1003 (2018).
Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285–289 (2021).
Li, L. et al. Single-cell multi-omics sequencing of human early embryos. Nat. Cell Biol. 20, 847–858 (2018).
Guo, F. et al. The transcriptome and DNA methylome landscapes of human primordial germ cells. Cell 161, 1437–1452 (2015).
Yan, L. et al. Single-cell RNA-seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131–1139 (2013).
Deglincerti, A. et al. Self-organization of the in vitro attached human embryo. Nature 533, 251–254 (2016).
Shahbazi, M. N. et al. Self-organization of the human embryo in the absence of maternal tissues. Nat. Cell Biol. 18, 700–708 (2016).
Zhou, F. et al. Reconstituting the transcriptome and DNA methylome landscapes of human implantation. Nature 572, 660–664 (2019).
Xiang, L. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537–542 (2020).
Wang, Y. et al. A single-cell characterization of human post-implantation embryos cultured in vitro delineates morphogenesis in primary syncytialization. Front. Cell Dev. Biol. 10, 835445 (2022).
Lv, B. et al. Single-cell RNA sequencing reveals regulatory mechanism for trophoblast cell-fate divergence in human peri-implantation conceptuses. PLoS Biol. 17, e3000187 (2019).
West, R. C. et al. Dynamics of trophoblast differentiation in peri-implantation-stage human embryos. Proc. Natl Acad. Sci. USA 116, 22635–22644 (2019).
Xu, Y. et al. A single-cell transcriptome atlas profiles early organogenesis in human embryos. Nat. Cell Biol. 25, 604–615 (2023).
Zeng, B. et al. The single-cell and spatial transcriptional landscape of human gastrulation and early brain development. Cell Stem Cell 30, 851–866.e7 (2023).
de Graeff, N., De Proost, L. & Munsie, M. ‘Ceci n’est pas un embryon?’ The ethics of human embryo model research. Nat. Methods 20, 1863–1867 (2023).
Nakamura, T., Fujiwara, K., Saitou, M. & Tsukiyama, T. Non-human primates as a model for human development. Stem Cell Rep. 16, 1093–1103 (2021).
Ma, H. et al. In vitro culture of cynomolgus monkey embryos beyond early gastrulation. Science 366, eaax7890 (2019).
Niu, Y. et al. Dissecting primate early post-implantation development using long-term in vitro embryo culture. Science 366, eaaw5754 (2019).
Zhai, J. et al. Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture. Cell 186, 2078–2091.e18 (2023).
Gong, Y. et al. Ex utero monkey embryogenesis from blastocyst to early organogenesis. Cell 186, 2092–2110.e23 (2023).
Hsu, Y. C. In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Dev. Biol. 68, 453–461 (1979).
Libbus, B. L. & Hsu, Y. C. Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage. Anat. Rec. 197, 317–329 (1980).
Chen, L. T. & Hsu, Y. C. Development of mouse embryos in vitro: preimplantation to the limb bud stage. Science 218, 66–68 (1982).
Hsu, Y. C. Differentiation in vitro of mouse embryos beyond the implantation stage. Nature 239, 200–202 (1972).
Bedzhov, I., Leung, C. Y., Bialecka, M. & Zernicka-Goetz, M. In vitro culture of mouse blastocysts beyond the implantation stages. Nat. Protoc. 9, 2732–2739 (2014).
Aguilera-Castrejon, A. et al. Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis. Nature 593, 119–124 (2021).
Gu, Z. et al. A uterus-inspired niche drives blastocyst development to the early organogenesis. Adv. Sci. 9, e2202282 (2022).
Yan, R. et al. Dynamics of DNA hydroxymethylation and methylation during mouse embryonic and germline development. Nat. Genet. 55, 130–143 (2023).
Kikkawa, T., Takahashi, M. & Osumi, N. Electroporation in the rodent embryonic brain using whole embryo culture system. Curr. Protoc. Neurosci. 78, 3 30 1–3 30 16 (2017).
Enders, A. C. Transition from lacunar to villous stage of implantation in the macaque, including establishment of the trophoblastic shell. Acta Anat. 152, 151–169 (1995).
Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. Cell 186, 3776–3792.e16 (2023).
Buse, E. & Markert, U. R. The immunology of the macaque placenta: a detailed analysis and critical comparison with the human placenta. Crit. Rev. Clin. Lab. Sci. 56, 118–145 (2019).
Ochoa-Bernal, M. A. & Fazleabas, A. T. Physiologic events of embryo implantation and decidualization in human and non-human primates. Int. J. Mol. Sci. 21, 1973 (2020).
Yoshimatsu, N. & Yanagimachi, R. Effects of cations and other medium components on the zona-induced acrosome reaction of hamster spermatozoa: (acrosome reaction/spermatozoa/cations/hamster). Dev. Growth Differ. 30, 651–659 (1988).
Bavister, B. D., Boatman, D. E., Leibfried, L., Loose, M. & Vernon, M. W. Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol. Reprod. 28, 983–999 (1983).
McKiernan, S. H. & Bavister, B. D. Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production. Hum. Reprod. 15, 157–164 (2000).
Takahashi, M., Makino, S., Kikkawa, T. & Osumi, N. Preparation of rat serum suitable for mammalian whole embryo culture. J. Vis. Exp. 3, e51969 (2014).
Rakvaag, E., Lund, M. D., Wiking, L., Hermansen, K. & Gregersen, S. Effects of different fasting durations on glucose and lipid metabolism in Sprague Dawley rats. Horm. Metab. Res. 51, 546–553 (2019).
Glanville-Jones, H. C., Woo, N. & Arkell, R. M. Successful whole embryo culture with commercially available reagents. Int. J. Dev. Biol. 57, 61–67 (2013).
Yang, S. et al. Effects of rhFSH dose on ovarian follicular response, oocyte recovery and embryo development in rhesus monkeys. Theriogenology 67, 1194–1201 (2007).
Piekarski, N. et al. A comparison of oocyte yield between ultrasound-guided and laparoscopic oocyte retrieval in rhesus macaques. Animals 13, 3017 (2023).
Baldini, D. et al. Sperm selection for ICSI: do we have a winner? Cells 10, 3566 (2021).
Nakamura, T. et al. A developmental coordinate of pluripotency among mice, monkeys and humans. Nature 537, 57–62 (2016).
Arakaki, P. R., Carvalho, F. M., Castro, P. H. G., Muniz, J. & Valle, R. Collection, evaluation, and coagulum dissolution of semen from Goeldi’s monkey, Callimico goeldii. Folia Primatol. 88, 334–343 (2017).
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2020YFA0112201, 2021YFA0805701, 2022YFA1103100), and the National Natural Science Foundation of China (NSFC, 82192870, 82322026, 32100654, 82201857, 82301883).
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H. Wang conceived and supervised the study. Y.X., J.Z. and H. Wu contributed equally to the study. Y.X., J.Z., H. Wu and H. Wang wrote the original draft and were involved in the editing and modification.
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Nature Protocols thanks Yasuhiro Takashima and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Zhai, J. et al. Cell 186, 2078–2091 (2023): https://doi.org/10.1016/j.cell.2023.04.019
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Xu, Y., Zhai, J., Wu, H. et al. In vitro culture of cynomolgus monkey embryos from blastocyst to early organogenesis. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01025-8
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DOI: https://doi.org/10.1038/s41596-024-01025-8
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