Stem Cell Analysis: Nucleic acid content

Although stem cells have a great capacity for self-renewal and their progeny have high proliferative capacity, it is thought that stem cells may only divide when necessary and for the most part may reside in a quiescent phase of the cell cycle. We have attempted to identify a population enriched for potential stem cells by looking for quiescent cells which:

• Have diploid DNA content
• Have low RNA content

There are fluorescent dyes that will quantitate DNA (eg Propidium iodide, DAPI, Hoechst) and dyes that will quantitate RNA content (Acridine Orange, Pyronin Y). We have used a combination of Hoechst 33342 (for DNA content) and Pyronin Y (for RNA content) to identify quiescent cells. Subsequent sorting of these cells shows that they have lower expression of the proliferation marker Ki-67.

The example below shows a population that is in the diploid phase of the cell cycle as judged by Hoechst uptake but in G0 as it has low Pyronin Y staining - this population is enriched for putative stem cells.