I am struggling to find information about this- I'm not sure if it is something so obvious that it is assumed rather than stated in reseach papers (!) or if it is just not the case, or maybe I am just looking in the wrong places!
I am trying out some differentiation protocols, and wondering if I can use a comparison of proliferation rates (for example with BrdU assay) between the 'control' stem cells (human foetal neural stem cells) and experimental cells to show they have differentiated
Any ideas gratefully received-
Thanks
Stem cells do have different proliferation rates, actually, they may not proliferate unless it is not necessary while the rest of the cells of the tissue keep proliferating. However, that not necessarily mean they proliferate slower than a normal cell line. I believe you need to consider other strategies to evaluate differentiation. I would study differentiation as loss of stemness. For example you can evaluate clonogenic potential of cells you suspect they have differentiated, you can extract RNA and look for expression of genes associated with stemness (sox9 for example). I used to work with breast cancer stem cells so I studied mammosphere forming capacity, you can check if your cells form tumorspheres of other structures related to stem cells with B27.
Check if my paper gives you some ideas:
https://www.researchgate.net/publication/311663715_Laminin_Modulates_the_Stem_Cell_Population_in_LM05-E_Murine_Breast_Cancer_Cells_through_the_Activation_of_the_MAPKERK_Pathway
Thanks Diego, that's good to know!
Just a bit more detail on my work:
I am also working with glioblastoma-derived cancer stem cells as well as the 'healthy' neural stem cells. In my previous experiments I have used PCR, qPCR, Western blot and immunostaining. I was considering looking into the proliferation as a sort of extra level of comparison- I'm also just quite curious!
As a side note, I noticed in your paper that you used qPCR to investigate loss of stemness- some of my qPCR data from previous experiments have shown an increase/ no change in stem cell markers (e.g SOX2/ Nestin) whilst also showing an increase in differentiation markers. Did you come across this at all, especially with cancer stem cells?
Sorry I just realised you did RT-PCR not qPCR in that particular paper
If you are working neural cancer stem cells, check if your cells make neurospheres, that can be a good and simple strategy to study stem cells. In theory each sphere comes from a cell with stem cell properties, that means that if stem are differentiating the sphere forming capacity of those cells is going to be reduced. It is a very simple and effective assay. Clonogenic assays will also give information of the proportion of stem cells in your cells line.
In regards to our paper, we saw decrease expression of genes related with stemness only. We did not study genes associated with differentiation, we were studding loss of stemness, we didn’t study to what those cells differentiate. I do remember however, that in a human cell line (MCF7), despite we saw reduction of mammosphere forming capacity and clonogenic potential, PCR results were not as clear as they were on the mouse model.
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