Human pluripotent stem cells (hPSCs) possess the unique ability to undergo the processes of self-renewal or differentiation into all somatic cell types derived from the embryonic germ layers: endoderm, mesoderm and ectoderm. hPSC potency can be evaluated using a number of strategies, of which the teratoma assay is the most widely-documented (Damjanov & Andrews, 2016). In this procedure, undifferentiated hPSCs are injected into an immunodeficient mouse, and develop into a heterogeneous tumor composed of terminally differentiated cells representative of all three germ layers. Unfortunately, this procedure can be lengthy, cost prohibitive and there is little consistency in the methodologies and analyses used (Bouma et al., 2017). As a result, alternative approaches to analyze hPSC potency are now being adopted.

When cultured in suspension, hPSCs spontaneously differentiate to form three-dimensional aggregates called embryoid bodies (EBs) (Itskovitz-Eldor et al., 2000). These structures recapitulate many features of cell differentiation during early embryogenesis and can display evidence of trilineage differentiation potential. Unfortunately, the EB assay has been difficult to standardize. Advances in protocols describing directed differentiation towards endoderm (D’Amour et al., 2005), mesoderm (Zhang et al., 2008) and ectoderm (Chambers et al., 2009) have provided researchers with the ease of use and specificity required in order to rapidly assess pluripotency.

It is important to note that common hPSC markers should be expressed by undifferentiated hPSCs, but are not necessarily markers of “pluripotency.” As a result, it is essential to go beyond measuring gene expression and validate the pluripotency of a hPSC line in a functional manner.

Key Publications

• This paper describes a highly efficient neural induction protocol using two inhibitors of SMAD signaling, Noggin and SB431542. Neural conversion of hPSCs is achieved using monolayer culture conditions as opposed to embryoid body formation.
  Chambers SM et al. (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27(3): 275-80.
• D’Amour et al. describe an efficient protocol for the directed differentiation of hES cells to definitive endoderm. Homogeneous differentiation was achieved by the addition of activin A in low-serum conditions followed by CXCR4 enrichment.
  D’Amour K et al. (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23: 1534-41.
• This atlas illustrates the microscopic features of tumors, or teratomas, produced from hPSCs xenografted into immunosuppressed mice.
  Damjanov I & Andrews PW (2016) Teratomas produced from human pluripotent stem cells xenografted into immunodeficient mice - a histopathology atlas. Int J Dev Biol 60(10-11-12): 337-419.
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• Itskovitz-Eldor et al. report the three-dimensional EB formation of human embryonic stem cells cultured in suspension, and their subsequent spontaneous differentiation to the three germ layers.
  Itskovitz-Eldor J et al. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6(2): 88-95.
• This paper shows an example of directed differentiation to mesoderm using BMP4. Mesoderm is induced with high efficiency and the cells can further differentiate into multiple mesodermal cell types.
  Zhang P et al. (2008) Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood 111(4): 1933-41.
• Bouma et al. compare in vivo teratoma and in vitro directed differentiation methods to assess pluripotency. They found that both methods confirmed pluripotency, with the in vitro method being much quicker. However, only the teratoma assay detected malignant potential. These results were also compared with the PluriTest gene expression analysis, which failed to distinguish between lines shown functionally to be differentiation-defective.
  Bouma MJ et al. (2017) Differentiation-Defective Human Induced Pluripotent Stem Cells Reveal Strengths and Limitations of the Teratoma Assay and In Vitro Pluripotency Assays. Stem Cell Reports 8(5): 1340-53.
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