I am trying to separated stem cells from old differentiated senescent to enrich a population of my culture. Stem cells are usually in a quiescent state, so to separate would be ideal for me.
By definition, quiescent cells can enter cell cycle upon providing a stimulus such as growth factors. In contrast, senescent cells are resistant to any stimulus and do not undergo cell division. Therefore, a stimulus to quiescent cells should resume cell cycle progression and cell division. These dividing cells can be separated from non-dividing cells by a number of approaches.
Thank you Dr. Choubey. I have read about this approach, but I have been unsuccessful with finding the right assay/technique to create a pure population of stem cells. I have several cultures, however I have had several instances of contamination of senescent cells. Do you recommend any specific procedure/assay please? Your assistance is much appreciated.
It may be more feasible to separate stem cells from their differentiated progeny as side population (SP) by FACS, using the appropriate dyes such as Hoechst 33342 or better DyeCycle Violet. Stem cells that over-express ABC transporters efflux these DNA-binding dyes, while differentiated cells retain them and display fluorescence corresponding to DNA-dye complexes.
Senescent cells may be separated from other cell populations by FACS based on their expression of senescence-associated beta-galactosidase (e.g. using the live cell analysis kit provided by Enzo Life Sciences).
It's hard to know if this advice is any good without understanding the type of tissue you are starting with, how you isolate your stem cells, and how you differentiate between the cells you want and the senescent ones. However, I wonder if you aren't isolating senescent cells, but instead they are becoming senescent once in culture. In this case, one possibility is to grow the cells in a low oxygen incubator. For fibroblasts at least, this has been shown to delay the onset of senescence. If you are starting with mice, a simple suggestion is to start with the youngest mice you can get. In the case of HSCs, this reduces the frequency of senescent ones. Finally, you mention "old differentiated senescent" cells as the problem. Terminally differentiated cells don't divide, but generally aren't called senescent. However, differentiated cells should express a variety of proteins absent from stem cells. If the problematic cells have lineage specific surface markers, you could remove them with antibodies and magnetic beads (such as the MACS system). Good luck!
Hello Dr. Givon Sanati,
One way to identify senescent cells is the evaluation of senescence-associated-B-galactosidase activity. There´s a Nature Protocol for its determination. I think that you could do a cell sorting by flow cytometry using CD34 marker (to see Stem Cells) and C12FDG molecular probe (to identify senescent cells). Good Luck!
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