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Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos

Nature Protocols volume 3, pages 923–933 (2008)Cite this article

Abstract

Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos that are discarded during the IVF procedure because of poor morphology and a low likelihood for generating viable pregnancies or surviving the cryopreservation process are also a viable source of hES cells. In this protocol, we describe how to derive novel hES cells from discarded poor-quality embryos and how to maintain the hES cell lines.

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Figure 1: Mechanical passaging of hES cell colony visualized with stereomicroscope (darkfield).
Figure 2: Collagenase treatment of hES cell colonies visualized with stereomicroscope (darkfield).
Figure 3: Human embryonic stem cell surface marker and oct3/4 expression of a hES cell line derived from a poor-quality embryo.
Figure 4: Histopathology of tumors resulting from in vitro differentiation assay using hES cell lines derived from poor-quality embryos.

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Acknowledgements

This work was made possible through the generosity and vision of Joshua and Anita Bekenstein, the Harvard Stem Cell Institute and Children's Hospital Boston. G.Q.D. is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

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Author notes

  1. Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.

Authors and Affiliations

  1. Division of Newborn Medicine, Brigham & Women's Hospital and Children's Hospital Boston, Karp Family Research Building 7214, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Paul H Lerou

  2. Division of Pediatric Hematology Oncology, Children's Hospital Boston, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Akiko Yabuuchi, Hongguang Huo, Justine D Miller, Leah F Boyer, Thorsten M Schlaeger & George Q Daley

  3. Harvard Stem Cell Institute, hES Cell Core Facility, Children's Hospital Boston, Karp Family Research Building 09213, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Hongguang Huo, Justine D Miller, Leah F Boyer, Thorsten M Schlaeger & George Q Daley

  4. Division of Hematology, Brigham and Women's Hospital, Karp Family Research Building 7214, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    George Q Daley

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Karp Family Research Building 7214, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    George Q Daley

  6. Harvard Stem Cell Institute, Karp Family Research Building 7214, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    George Q Daley

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  1. Paul H Lerou
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Contributions

P.H.L. and A.Y. contributed equally to this work.

Corresponding author

Correspondence to George Q Daley.

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Lerou, P., Yabuuchi, A., Huo, H. et al. Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos. Nat Protoc 3, 923–933 (2008). https://doi.org/10.1038/nprot.2008.60

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