[English]  [Pусский]  [中文]  
 
ctt-journal > Maximow Award > Blog Comments > Comment 11

Comment 11

Please cite this contribution as follows: Alexey [Bersenev]. Ilya, Colony assay is not assessing stem cells, it’s. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_11

This contribution is provided under the following license: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

pdf
Contribute a comment


Alexey. June 1, 2012 at 3:52 pm

Ilya,
Colony assay is not assessing stem cells, it’s assessing progenitors, unless you do single cell. But, even if you put a single cell sorted as “HSC” you don’t know is it a real stem cell or not without transplantation. Markers are not always translate to function. Remember, in the best case, 1 out 4 sorted cells will repopulate a mouse. So, we never use colony assay to make conclusion about HSC. Culture (in vitro) is very much artificial system and alter HSC function. That’s why golden standard to assess HSC (self-renewal and multilineage differentiation) is in vivo (transplant) assays.

How would you assess self-renewal in colony assay? The most sophisticated method could include seeding of single cell, get a colony 1 week after, replate this colony (or single stem cell from this colony) to other well and get secondary colony 1 week later. Do a few cycles of re-plating until HSC exhaust. But currently nobody is doing that, because transplantation assays are more trusted and commonly accepted. So if you will try to “sell your story” about HSC function based solely on colony assay, you will never able to publish it. Any reviewer will ask for transplantation assay. Colony assay could be used only as an additional (to transplantation) method to confirm progenitors performance. Transplantation assays can cover absolutely all possibilities on colony assay. You can check any lineage maker, derived from donor’s HSC, you can measure self-renewal nd quantify HSC frequency.

The reason why Morita did it, was not HSC self-renewal assessment, but rather additional method to confirm/ reject in vivo data. In fact, they didn’t check Meg/erythro- repopulation in translant assay and they did so in colony assay.

To your second point. You don’t need to do single cell transplantation to assess HSC function. So, we frequently don’t do single cell. I personaly done 3, 10, 30, 100 sorted cells or 3k, 10k, 20k, 50k, 100k, 200k, 0.5M, 1M, 2M, 5M, 10M, 20M whole marrow. I referred only to single cell transplant studies, because we were discussing the problem of HSC heterogeneity. You can nail it down only on single cell level. Because I don’t study HSC heterogeneity, I don’t do single cell transplants. Practically you sort cells in each well of 96-well plate, control cell presence under the microscope, mix them with 0.2M or fresh competitor cells and transplant IV. Single HSC are functional, do migrate, engraft and repopulate a mouse. You can read the protocols in studies done by groups I mentioned earlier.

I still don’t understand what do you mean by “Clonal analysis allows us to manipulate a single cell, but serial transplantation does not allow to do it.” As I mentioned many times we can do any thing by single cell transplant and serial transplant. We can also play with single cell in vitro, but possibilities is limited and data should be interpreted with caution.

About the practicallity of deviding HSC for subpopulations. Frequently it doesn’t really matter for clinicians and medical points of view. Heterogeneity was discovered as biological phenomenon. So, now we know it exists. In clinical HSC transplantation we don’t need to dissect this heterogeneity, because we always transplant enriched HSC/ progenitor cell population or total mononuclear cells. A lot of basic science discoveries have a little clinical significance. For example, why clinician need to know how many defined progenitors exist between common lymphoid progenitor cell and mature NK cell? Maybe 3 maybe 5 maybe 7? Still a lot of scientists continue to work on this and get government fundings. I’m all for clinical relevance.
The clinical significance of HSC heterogeneity cold come from comparing normal HSC with leukemic SC. If it turns out that leukemic SC are also heterogenous, we should adjust our treatment strategies. For example, the current hypothesis says that leukemic SC are quiescent and to target them we need to make them cycle. But if we extrapolate normal HSC data, we can realize that leukemic SC could switch quiescence to cycling back and forth and become undruggable. The first thing which came up recently about leukemic SC heterogeneity is a great variability or surface markers expression. They can turn them off and on all the time. So, the clinical significance of leukemic SC heterogeneity is that we can’t use antibody to surface markers to target them selectively.

Finally, about the mice. Genetically identical means the same breed. For example, C57B6J or SJL as recipients. When you do thousand of transplants and you don’t see a difference between all repopulation in normal conditions, you can realize and appreciate how remarkable similar they are and how model is “clean”. Of course, unless you start to titrate cell doses down to single cell. That’s how the whole heterogeneity story unfolded.

This contribution is provided under the following license: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

Please cite this contribution as follows: Alexey [Bersenev]. Ilya, Colony assay is not assessing stem cells, it’s. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_11

All comments