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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

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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Figure 1: Morphology of spin embryoid bodies (EBs) generated in APEL and BPEL media.
Figure 2: Generation of spin embryoid bodies (EBs) from human embryonic stem cells (HESCs).
Figure 3: Spin embryoid bodies (EBs) generated in APEL and BPEL media supplemented with bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF) and stem cell factor (SCF) form hematopoietic mesoderm.
Figure 4: Spin embryoid bodies (EBs) generated in BPEL media with different ratios of BSA/PVA supplemented with bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF) and stem cell factor (SCF) form equivalent amounts of mesoderm.

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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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Correspondence to Andrew G Elefanty.

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Ng, E., Davis, R., Stanley, E. et al. 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. Nat Protoc 3, 768–776 (2008). https://doi.org/10.1038/nprot.2008.42

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