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Derivation of human embryonic stem cells using a post–inner cell mass intermediate

Nature Protocols volume 8, pages 254–264 (2013)Cite this article

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Abstract

Little is known about the true developmental origin of human embryonic stem cells (hESCs) or the events that initiate their generation. Recently, we have shown that hESCs originate from a post–inner cell mass (ICM) intermediate (PICMI), a unique transient epiblast-like structure that is different from both its ICM progenitor and its subsequent hESC fate. As a closer progenitor of hESCs than the ICM, the PICMI could be used to provide further insight into the human pluripotent state. Here we provide a detailed (7-d) protocol for the culture of the human preimplantation embryos in order to derive the PICMI. Subsequent identification and cryopreservation of the PICMI are described, in addition to hESC derivation. The initial hESC outgrowth is visible within 2–7 d after PICMI plating. By using the protocol provided, we observed PICMI formation in 21.3% of plated blastocysts with good-quality ICMs. Of the PICMIs used for hESC derivation, 80.6% showed hESC outgrowth after further culture.

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Figure 1: Grading human preimplantation embryos.
Figure 2: PICMI development in culture.
Figure 3: PICMI morphology in culture.
Figure 4: PICMI vitrification.

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Acknowledgements

This work was supported by the Flemish Foundation for Scientific Research (FWO-Vlaanderen (grant no. FWO-3G062910) and by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds to P.D.S. (grant no. BOF GOA 01G01112). S.M.C.S.L. was supported by the Netherlands organization of Scientific Research (NWO) (no. ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P6/20). P.D.S. is the holder of a fundamental clinical research mandate by the Flemish Foundation for Scientific Research (FWO-Vlaanderen).

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Author notes

  1. Petra De Sutter and Susana M Chuva de Sousa Lopes: These authors contributed equally to this work.

Authors and Affiliations

  1. Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium

    Thomas O'Leary, Björn Heindryckx, Sylvie Lierman, Margot Van der Jeught, Galbha Duggal & Petra De Sutter

  2. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands

    Susana M Chuva de Sousa Lopes

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Contributions

T.O., B.H., P.D.S. and S.M.C.d.S.L. designed the study; T.O., S.L., M.V.d.J. and G.D. performed the experiments; and T.O., B.H., S.L. and S.M.C.d.S.L. analyzed the data and wrote the manuscript.

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Correspondence to Thomas O'Leary.

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O'Leary, T., Heindryckx, B., Lierman, S. et al. Derivation of human embryonic stem cells using a post–inner cell mass intermediate. Nat Protoc 8, 254–264 (2013). https://doi.org/10.1038/nprot.2012.157

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