Pluripotency Assay

Functional pluripotency can only be assessed through differentiation of human pluripotent stem cells (hPSCs) and evaluation of marker expression specific to each of the three germ layers, endoderm, mesoderm and ectoderm.  Until recently, “gold standard” to assess pluripotency of a cell line was teratoma formation using immune deficient (SCID-Beige) mice. However, a recent publication by The International Stem Cell Initiative (ISCI), a consortium of select stem cell researchers led by Dr. Peter Andrews (Sheffield University, Sheffield, UK) and Dr. Martin Pera (Jackson Labs, Bar Harbor, ME) identified more feasible alternatives that can be used to clearly demonstrate pluripotency (Allison, 2018). To assess pluripotency, WiCell uses the StemDiff™ Trilineage Differentiation Kit to directly differentiate cell cultures to the three germ lineages - endoderm, ectoderm and mesoderm.  Flow cytometry is then used to assess the expression of   markers specific for each of the three germ layers.  Specific Markers are as follows:

  • Endoderm:  Dual positive expression of Sox 17 and CXCR4
  • Mesoderm:  Dual positive expression of Brachyury and NCAM
  • Ectoderm:  Dual positive expression of PAX 6 and Nestin

Expression as described in any lineage confirms the ability to differentiate into that specific lineage.  Expression or markers in al three lineages (endoderm, mesoderm and ectoderm) confirms the pluripotency of a given cell culture, and can be used as a reflective assessment of the pluripotent potential of the cell line in general.  

Interpreting your results

When your assay is complete you will receive a Assessment of Pluripotency Report.  Please see our sample Assessment of Pluripotency report. 


International Stem Cell Initiative; Allison, T. et al., Assessment of established techniques to determine developmental and malignant potential of human pluripotent stem cells." 2018.  Nature Com. 9: 1925. doi:10.1038/s41467-018-04011-3