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A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies

Nature Protocols volume 3, pages 768776 (2008) | Download Citation

Abstract

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

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References

  1. 1.

    , , , & Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 97, 11307–11312 (2000).

  2. 2.

    et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102, 906–915 (2003).

  3. 3.

    et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

  4. 4.

    et al. In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res. 17, 333–344 (2007).

  5. 5.

    , , , & Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 98, 10716–10721 (2001).

  6. 6.

    , & Slukvin, II Human embryonic stem cell-derived CD34. cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105, 617–626 (2005).

  7. 7.

    , , , & Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).

  8. 8.

    et al. Generation of functional hemangioblasts from human embryonic stem cells. Nat. Methods 4, 501–509 (2007).

  9. 9.

    , , , & Large-scale production of embryonic red blood cells from human embryonic stem cells. Exp. Hematol. 34, 1635–1642 (2006).

  10. 10.

    et al. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat. Med. 13, 642–648 (2007).

  11. 11.

    et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392–1401 (2006).

  12. 12.

    et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25, 4694–4705 (2005).

  13. 13.

    et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 111, 1876–1884 (2008).

  14. 14.

    , , , & Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106, 1601–1603 (2005).

  15. 15.

    , , , & Differentiation of human embryonic stem cells in serum free medium reveals distinct roles for BMP4, VEGF, SCF and FGF2 in hematopoiesis. Stem Cells 25, 2206–2214 (2007).

  16. 16.

    et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792–796 (2007).

  17. 17.

    & Evidence for involvement of Activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoiesis development. Mol. Cell Biol. 15, 141–151 (1995).

  18. 18.

    , , & Culture of colony-forming hematopoietic progenitor cells from human peripheral blood. J. Tissue Cult. Metheds 14, 13–20 (1992).

  19. 19.

    et al. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat. Methods 2, 259–260 (2005).

  20. 20.

    et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells 25, 929–938 (2007).

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Acknowledgements

This work was supported by the Australian Stem Cell Centre and the National Health and Medical Research Council of Australia (NHMRC). A.G.E. is a Senior Research Fellow of the NHMRC.

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Affiliations

  1. Monash Immunology and Stem Cell Laboratories, STRIP 1, Building 75, Level 3, Monash University, Clayton, Victoria 3800, Australia.

    • Elizabeth S Ng
    • , Richard Davis
    • , Edouard G Stanley
    •  & Andrew G Elefanty

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Correspondence to Andrew G Elefanty.

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https://doi.org/10.1038/nprot.2008.42

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