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Generating human blastoids modeling blastocyst-stage embryos and implantation

Nature Protocols volume 18, pages 1584–1620 (2023)Cite this article

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Abstract

Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into structures resembling preimplantation embryos—blastocysts—that we termed blastoids. This embryo model is available in unlimited numbers and could thus support scientific and medical advances. However, its predictive power depends on how faithfully it recapitulates the blastocyst. Here, we describe how we formed human blastoids that (1) efficiently achieve the morphology of the blastocyst and (2) form lineages according to the pace and sequence of blastocyst development, (3) ultimately forming cells that transcriptionally reflect the blastocyst (preimplantation stage). We employ three different commercially available 96- and 24-well microwell plates with results similar to our custom-made ones, and show that blastoids form in clinical in vitro fertilization medium and can be cryopreserved for shipping. Finally, we explain how blastoids replicate the directional process of implantation into endometrial organoids, specifically when these are hormonally stimulated. It takes 4 d for human blastoids to form and 10 d to prepare the endometrial implantation assay, and we have cultured blastoids up to 6 d (time-equivalent of day 13). On the basis of our experience, we anticipate that a person with ~1 year of human pluripotent stem cell culture experience and of organoid culture should be able to perform the protocol. Altogether, blastoids offer an opportunity to establish scientific and biomedical discovery programs for early pregnancy, and an ethical alternative to the use of embryos.

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Fig. 1: A schematic of the time window of human peri-implantation development modeled by blastoids.
Fig. 2: Schematic overview of the protocol.
Fig. 3: Four basic criteria to validate the formation of blastoids.
Fig. 4: Morphological characterization of PXGL hPSCs.
Fig. 5: Triple inhibition of Hippo, ERK and TGFβ pathways leads to efficient and robust formation of human blastoids composed of blastocyst-like cells.
Fig. 6: Human blastoids resemble the morphology of the blastocyst, comprise analogs of the three founding lineages and form an axis.
Fig. 7: Representative images of endometrial organoids and monolayers.
Fig. 8: Human blastoids recapitulate aspects of implantation.

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Data availability

The main data discussed in this protocol were generated as part of the study published in the supporting primary research paper by Kagawa et al.26. Representative results obtained using this protocol are available within the article, with additional examples available from the corresponding author upon request.

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Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-Co grant agreement no. 101002317 ‘BLASTOID: a discovery platform for early human embryogenesis’). This project has received funding from the Austrian Science Fund (FWF), Lise Meitner Programme M3131-B, H.H.K. is supported by FWF. This project has received funding from the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014–2020) under the Marie Skłodowska-Curi grant agreement no. 101026451. H.K. is supported by the Japan Society for the Promotion of Science Overseas Research Fellowships. G.S. is supported by the HFSP number RGY0081/2019. We thank Y. Takashima for sharing the H9 and H9-GFP cell lines; A. Smith, P. Andrews and G. Guo for sharing the HNES1, Shef6, niPSC 16.2b and cR-NCRM2 cell lines; H. Baharvand for sharing the endometrial organoids; S. Srinivas for sharing the scRNA-seq data of peri-gastrulation embryo; A. Bykov and L. Cochella for technical assistance for SMARTSeq2 library preparation; and the NGS, Biooptic and Stem Cell facility at IMBA for critical assistance.

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Author notes

  1. These authors contributed equally: Heidar Heidari Khoei, Alok Javali, Harunobu Kagawa.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria

    Heidar Heidari Khoei, Alok Javali, Harunobu Kagawa, Theresa Maria Sommer, Giovanni Sestini, Maria Novatchkova, Yvonne Scholte op Reimer & Nicolas Rivron

  2. Université de Nantes, CHU Nantes, Inserm, CR2TI, UMR 1064, Nantes, France

    Laurent David

  3. Université de Nantes, CHU Nantes, Inserm, CNRS, BioCore, Nantes, France

    Laurent David

  4. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), IMBA Stem Cell Core Facility (ISCCF), Vienna BioCenter (VBC), Vienna, Austria

    Jana Slovakova

  5. Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria

    Maria Novatchkova

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  1. Heidar Heidari Khoei
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Contributions

H.H.K., A.J., H.K. and N.R. conceived the study; N.R. supervised the project; H.K., A.J., H.H.K., T.M.S. and N.R. designed the blastoid experiments; H.K., A.J., H.H.K., T.M.S., J.S. and Y.S.o.R. performed blastoid experiments; G.S., L.D. and M.N. performed the bioinformatic analysis of scRNA-seq datasets; H.K., A.J., H.H.K., T.M.S., Y.S.o.R. and N.R. analyzed data; N.R. wrote the manuscript with help from all the authors.

Corresponding author

Correspondence to Nicolas Rivron.

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Competing interests

The Institute for Molecular Biotechnology, Austrian Academy of Sciences has filed patent application EP21151455.9 describing the protocols for human blastoid formation and for the blastoid–endometrium interaction assay. H.K., A.J., H.H.K. and N.R. are the inventors on this patent. All other authors declare no competing interests.

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Nature Protocols thanks Susana Chuva de Sousa Lopes, Kiichiro Tomoda and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key paper using this protocol

Kagawa, H. et al. Nature 601, 600–605 (2022): https://doi.org/10.1038/s41586-021-04267-8

Extended data

Extended Data Fig. 1 Triple inhibition of Hippo, ERK and TGFβ pathways leads to efficient and robust formation of human blastoids.

Time course bright-field images of PXGL hPSCs aggregates and blastoid formation within AggreWell (top) and microwell arrays (bottom) in PALLY medium. Scale bars, 400 μm.

Extended Data Fig. 2 Human blastoids formation in IVF medium and vitrification.

a, Bright-field image of human blastoids formed after 48 h stimulation with PALLY medium followed by the use of IVF medium (G2, Vitrolife) for the last 2 d. Scale bars, 400 µm. b, Bright-field image of control (top) and vitrified-thawed human blastoid (bottom) and after 2 d extended culture on Matrigel-coated plate. Scale bars, 100 µm. c, Confocal immunofluorescence image of OCT4 (yellow) and aPKC (gray) in control (top) and vitrified-thawed human blastoid (bottom) cultured on Matrigel-coated plate for 2 d, counterstained with phalloidin marking F-actin (cyan). Arrows point to the pro-amniotic-like cavity. Scale bar, 100 μm.

Supplementary information

Supplementary Information

Supplementary Tables 1and 2.

Supplementary Video 1

Human blastoids attach via the polar trophectoderm. Live imaging of human blastoids attached via the polar region to hormonally stimulated layers of endometrial epithelial cells and challenged by pipetting liquid in their vicinity.

Supplementary Video 2

Human blastoids on non-hormonally stimulated endometrial layers. Live imaging of human blastoids on non-hormonally stimulated layers of endometrial epithelial cells, and challenged by pipetting liquid in their vicinity. Blastoids do not show attachment.

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Heidari Khoei, H., Javali, A., Kagawa, H. et al. Generating human blastoids modeling blastocyst-stage embryos and implantation. Nat Protoc 18, 1584–1620 (2023). https://doi.org/10.1038/s41596-023-00802-1

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