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Derivation of human embryonic stem cells from single blastomeres

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Abstract

This protocol details a method to derive human embryonic stem (hES) cells from single blastomeres. Blastomeres are removed from morula (eight-cell)-stage embryos and cultured until they form multicell aggregates. These blastomere-derived cell aggregates are plated into microdrops seeded with mitotically inactivated feeder cells, and then connected with neighboring microdrops seeded with green fluorescent protein-positive hES cells. The resulting blastomere-derived outgrowths are cultured in the same manner as blastocyst-derived hES cells. The whole process takes about 3–4 months.

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Figure 1: Examples of blastomere development before plating.
Figure 2: Plate setup for culture of blastomere-derived cell aggregates.
Figure 3: Examples of outgrowth after plating onto PMEFs.

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Acknowledgements

We thank Tong Li and Marc Maserati for help with cell culture, karyotyping and embryo manipulations.

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Authors and Affiliations

  1. Advanced Cell Technology, 381 Plantation Street, Worcester, 01605, Massachusetts, USA

    Irina Klimanskaya, Young Chung, Sandy Becker, Shi-Jiang Lu & Robert Lanza

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  1. Irina Klimanskaya
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  2. Young Chung
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  3. Sandy Becker
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  4. Shi-Jiang Lu
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  5. Robert Lanza
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Correspondence to Robert Lanza.

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The authors are employees of Advanced Cell Technology, a biotechnology company in the field of stem cell research and regenerative medicine.

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Klimanskaya, I., Chung, Y., Becker, S. et al. Derivation of human embryonic stem cells from single blastomeres. Nat Protoc 2, 1963–1972 (2007). https://doi.org/10.1038/nprot.2007.274

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