Abstract
The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development.
Key points
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This protocol describes the generation of induced blastoids, an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This model overcomes restrictions associated with the use of human blastocysts in embryology research.
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Compared with other blastoid models, which are generated from established stem cell lines, this protocol leverages on the cellular plasticity in the reprogramming cells, allowing direct generation of blastocyst-like structures from fibroblast.
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Data availability
The main data discussed in this protocol were generated as part of the studies published in the supporting primary research paper18.
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Acknowledgements
The authors acknowledge the use of the services and facilities of Monash Micro Imaging at Monash University. This work was supported by the National Health and Medical Research Council project grants APP1104560 and APP2004627 as well as a Synergy grant 2019251 to J.M.P. X.L. was supported by Westlake Education Foundation. J.P.T. was supported by a Monash International Tuition Scholarship, Research Training Program Scholarship and Carmela and Carmelo Ridolfo Prize in Stem Cell Research. J.M.P. was supported by an Australian Research Council Future Fellowship. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. The South Australian immunoGENomics Cancer Institute received grant funding from the Australian Government.
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Contributions
J.P.T. and X.L. drafted the protocol. J.P.T., X.L. and J.M.P. wrote the protocol. Most experiments presented in this protocol were performed by J.P.T. with supervision from X.L. and J.M.P.
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Competing interests
J.P.T, X.L. and J.M.P. are co-inventors on two Patent Cooperation Treaty (PCT) patent applications (application numbers PCT/AU2021/051401 and PCT/AU2021/051402) filed by Monash University related to work on derivation of human iBlastoids and derivatives. J.M.P. is a co-inventor on a patent (WO/2017/106932) and is a co-founder and shareholder of Mogrify Ltd., a cell therapy company. X.L is a co-founder of iCamuno Biotherapeutics Ltd. The other authors declare no competing interests.
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Nature Protocols thanks Yang Yu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Liu, X. et al. Nature 591, 627–632 (2021): https://doi.org/10.1038/s41586-021-03372-y
Key data used in this protocol
Liu, X. et al. Nature 586, 101–107 (2020): https://doi.org/10.1038/s41586-020-2734-6
Extended data
Supplementary information
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Tan, J.P., Liu, X. & Polo, J.M. Reprogramming fibroblast into human iBlastoids. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00984-2
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DOI: https://doi.org/10.1038/s41596-024-00984-2
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