AbstractThe embryonic and extraembryonic tissue interactions underlying human embryogenesis at implantation stages are not currently understood. We have generated a pluripotent stem cell-derived model that mimics aspects of peri-implantation development, allowing tractable experimentation otherwise impossible in the human embryo.

摘要目前尚不清楚植入阶段人类胚胎发生的胚胎和胚外组织相互作用。我们已经产生了一种多能干细胞衍生的模型，该模型模拟了植入周围发育的各个方面，从而可以进行易于处理的实验，否则在人类胚胎中是不可能的。

Activation of the extraembryonic lineage-specific transcription factors GATA6 and SOX17 (hypoblast factors) or GATA3 and TFAP2C (encoding AP2γ; trophoblast factors) in human embryonic stem (ES) cells drive conversion to extraembryonic-like cells. When combined with wild-type ES cells, self-organized embryo-like structures form in the absence of exogenous factors, termed human inducible embryoids (hiEmbryoids).

人胚胎干（ES）细胞中胚外谱系特异性转录因子GATA6和SOX17（成纤维细胞因子）或GATA3和TFAP2C（编码AP2γ；滋养层因子）的激活驱动向胚外样细胞的转化。当与野生型ES细胞结合时，在不存在外源因子的情况下形成自组织的胚胎样结构，称为人类诱导型胚状体（hiEmbryoids）。

The epiblast-like domain of hiEmbryoids polarizes and differentiates in response to extraembryonic-secreted extracellular matrix and morphogen cues. Extraembryonic mesenchyme, amnion and primordial germ cells are specified in hiEmbryoids in a stepwise fashion. After establishing stable inducible ES lines and converting ES cells to RSeT culture media, the protocol takes 7–10 d to generate hiEmbryoids.

hiEmbryoids的外胚层样结构域响应胚外分泌的细胞外基质和形态发生素线索而极化和分化。胚外间充质，羊膜和原始生殖细胞以逐步的方式在hiembryoid中指定。在建立稳定的诱导型ES细胞系并将ES细胞转化为RSeT培养基后，该方案需要7-10天才能产生hiembryoid。

Generation of hiEmbryoids can be performed by researchers with basic expertise in stem cell culture.Key points.

具有干细胞培养基本专业知识的研究人员可以进行hiembryoid的产生。关键点。

A protocol for the generation of a stem cell-derived human postimplantation embryo model by the combination of embryonic and transgene-induced extraembryonic-like cells. This model mimics aspects of peri-implantation development, allowing tractable experimentation otherwise impossible in the human embryo..

。该模型模拟了植入周围发育的各个方面，允许在人类胚胎中进行易于处理的实验。。

This model does not require an inductive pulse of signaling to drive differentiation and self-organization, allowing study of endogenous cues driving self-organization, differentiation and morphogenesis.

该模型不需要诱导信号脉冲来驱动分化和自组织，从而可以研究驱动自组织，分化和形态发生的内源性线索。

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Fig. 1: Overview of hiEmbryoid generation.Fig. 2: Efficient production of hiEmbryoids at a range of cell densities.Fig. 3: Example hiEmbryoids demonstrating correct organization.Fig. 4: Utility of hiEmbryoids to study morphogen signaling and differentiation in situ.

图1：hiEmbryoid生成概述。图2：在一系列细胞密度下有效生产hiembryoid。图3：展示正确组织结构的示例hiEmbryoids。图4:hiEmbryoids用于原位研究形态发生信号传导和分化。

Data availability

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Supporting data can be found in the supporting research article18 and are available from the corresponding author upon request.

支持数据可以在支持研究文章18中找到，并可根据要求从通讯作者那里获得。

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Download referencesAcknowledgementsThe authors thank members of the Zernicka-Goetz laboratory and Macarena Fuentes-Guajardo for helpful discussion and troubleshooting of the protocol, as well as J. Hanna and A. Brivanlou for providing cell lines. This work was supported by the Wellcome Trust (207415/Z/17/Z), the European Research Council (669198), the Allen Discovery Center for Lineage Tracing, Open Atlas and NOMIS award grants to M.Z.-G.

下载参考文献致谢作者感谢Zernicka Goetz实验室和Macarena Fuentes Guajardo的成员对该协议的有益讨论和故障排除，以及J.Hanna和A.Brivanlou提供的细胞系。这项工作得到了惠康信托基金（207415/Z/17/Z），欧洲研究理事会（669198），艾伦发现血统追踪中心，开放地图集和诺米斯奖授予M.Z.-G的支持。

C.W.G. was supported by the Leverhulme Trust. B.A.T.W. was supported by the Gates Cambridge Trust.Author informationAuthor notesThese authors contributed equally: Carlos W. Gantner, Bailey A. T. Weatherbee.Authors and AffiliationsMammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UKCarlos W.

C、 W.G.得到了Leverhulme Trust的支持。B、 A.T.W.得到了盖茨-剑桥信托基金会的支持。作者信息作者注意到这些作者做出了同样的贡献：Carlos W.Gantner，Bailey A.T.Weatherbee。作者和所属机构剑桥大学生理学，发育和神经科学系哺乳动物胚胎和干细胞小组，英国剑桥大学卡洛斯W。

Gantner, Bailey A. T. Weatherbee, Yuntao Wang & Magdalena Zernicka-GoetzStem Cell Embryo Models Group, Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USAMagdalena Zernicka-GoetzAuthorsCarlos W. GantnerView author publicationsYou can also search for this author in.

Gantner，Bailey A.T.Weatherbee，Yuntao Wang＆Magdalena Zernicka Goetz干细胞胚胎模型组，加利福尼亚理工学院生物与生物工程系，加利福尼亚州帕萨迪纳，美国Magdalena Zernicka Goetz作者Carlos W.GantnerView作者出版物您也可以在中搜索这位作者。

PubMed Google ScholarBailey A. T. WeatherbeeView author publicationsYou can also search for this author in

PubMed Google ScholarBailey A.T.WeatherbeeView作者出版物您也可以在

PubMed Google ScholarYuntao WangView author publicationsYou can also search for this author in

PubMed Google ScholarYuntao WangView作者出版物您也可以在

PubMed Google ScholarMagdalena Zernicka-GoetzView author publicationsYou can also search for this author in

PubMed Google ScholarMagdalena Zernicka GoetzView作者出版物您也可以在

PubMed Google ScholarContributionsC.W.G., B.A.T.W. and Y.W. performed experiments. C.W.G. and B.A.T.W. developed the protocol. M.Z.-G. supervised the project and provided reagents and funding. C.W.G. wrote the manuscript with input from B.A.T.W. and M.Z.-G.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献中心。W、 G.，B.A.T.W.和Y.W.进行了实验。C、 W.G.和B.A.T.W.制定了该协议。M、 Z.-G.监督该项目并提供试剂和资金。C、 W.G.在B.A.T.W.和M.Z.-G.的输入下撰写了手稿。相应的作者回复。

Magdalena Zernicka-Goetz.Ethics declarations

Magdalena Zernicka Goetz。道德宣言

Competing interests

相互竞争的利益

C.W.G., B.A.T.W. and M.Z.-G. are inventors on a patent application (number 63/403,684). Specific aspect of the manuscript covered in patent application: stem cell-derived model of the human embryo. M.Z.-G. holds several patent applications on the generation of stem cell-derived embryo models.

C、 W.G.、B.A.T.W.和M.Z.-G.是专利申请（编号63/403684）的发明人。专利申请中涉及的手稿的具体方面：干细胞衍生的人类胚胎模型。M、 Z.-G.拥有几项关于干细胞衍生胚胎模型生成的专利申请。

Peer review

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Peer review information

同行评审信息

Nature Protocols thanks Yue Shao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Nature Protocols感谢Yue Shao和另一位匿名审稿人为这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Related linksKey reference using this protocolWeatherbee, B. A. T. et al. Nature 622, 584–593 (2023): https://doi.org/10.1038/s41586-023-06368-yExtended dataExtended Data Fig.

Additional informationPublisher的注释Springer Nature在已发布地图和机构隶属关系中的管辖权主张方面保持中立。使用此协议的相关linksKey参考Weatherbee，B.A.T.等人，《自然》622584–593（2023）：https://doi.org/10.1038/s41586-023-06368-yExtended数据扩展数据图。

1 Example images of successful and unsuccessful hiEmbryoid experiments.a, Media overview for hiEmbryoid generation. b, Example brightfield images at days 1, 4 and 6 demonstrating side-by-side images of successful and unsuccessful hiEmbryoid generation. hiEmbryoids should show clear, rounded aggregation by day 1, indicative of high cell survival.

1成功和不成功的hiEmbryoid实验的示例图像。a，hiEmbryoid生成的媒体概述。b、 在第1天、第4天和第6天的示例明场图像显示了成功和不成功的hiEmbryoid生成的并排图像。到第1天，hiEmbryoids应该显示出清晰的圆形聚集，表明细胞存活率很高。

By day 4, hiEmbryoids show a rounded morphology with organized inside–outside morphology. hiEmbryoids picked at day 4 can be cultured further in individual 96 wells and maintain a clear inner, epithelial domain. Scale bars, 400 µm.Extended Data Fig. 2 Plasmid maps for generation of inducible cell lines.a–d.

到第4天，hiEmbryoids表现出圆形形态，内部-外部形态有序。在第4天挑选的hiembryoid可以在单个96孔中进一步培养，并保持清晰的内部上皮结构域。比例尺，400微米。。

TetO destination plasmids used to transfect human ES cells to enable doxycycline-inducible transgene expression for GATA6 (a), SOX17 (b), TFAP2C (c) and GATA3 (d). Pbase and rtTA plasmids were cotransfected to enable stable integration and expression of the Tet transactivator. Note that not all features are shown in the plasmid map.Rights and permissionsReprints and permissionsAbout this articleCite this articleGantner, C.W., Weatherbee, B.A.T., Wang, Y.

用于转染人ES细胞的TetO目的质粒，以使多西环素诱导的GATA6（a），SOX17（b），TFAP2C（c）和GATA3（d）转基因表达成为可能。共转染Pbase和rtTA质粒，以实现Tet反式激活因子的稳定整合和表达。请注意，并非所有特征都显示在质粒图中。权利和许可本文引用本文Gantner，C.W.，Weatherbee，B.A.T.，Wang，Y。

et al. Assembly of a stem cell-derived human postimplantation embryo model..

等。干细胞衍生的人类植入后胚胎模型的组装。。

Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01042-7Download citationReceived: 18 October 2023Accepted: 24 June 2024Published: 11 September 2024DOI: https://doi.org/10.1038/s41596-024-01042-7Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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