I am hoping you can help with a verification issue. We are currently using telomerase to identify any undifferentiated PSCs. We would like to add a second method for verification purposes. Which method do you think is best?

Answer

Elimination of pluripotent stem cells from differentiated cells is critical prior to downstream in vivo experiments. The most simple method would involve using bead-conjugated antibodies against the pluripotent surface marker SSEA4 (see link below). http://www.lifetechnologies.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/cell-separation-methods/embryonic-stem-cell-isolation-protocols/dynabeads-ssea-4.html

Using flow cytometry, SSEA4 negative population can be further assessed for the presence of cells expressing SSEA4 and other pluripotent markers such as TRA-1-60 and TRA-1-180.

Recently, alternate methods are emerging that are summarized in a recently publication in Nature Protocols {Ben-David & Benvenisty Nature Protocols (2014) 9, 729-740}.