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?

This question is part of the following Ask The Expert session:

Options for Identification and Characterization of Pluripotent Stem Cells

Answered by:

Dr Uma Lakshmipathy

Company: Thermo Fisher Scientific, Inc.

Job Title: Principal Scientist

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}.

Previous Question:

We have been working to reduce serum in our stem cell culture while maintaining pluripotent characteristics. Our standard media contains 10% FBS and we have not been able to reduce this amount successfully without the culture suffering. We recently were able to use human serum instead at 1.25% while maintaining similar pluripotent characteristics. However, we have concerns about adopting a process that uses human serum in a potential human therapy. Do you have any recommendations on supplements that might work to help us reduce the FBS down to a similar level as what we have achieved when using human serum?

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