Tracking divisional history of hematopoietic stem cells

by Alexey Bersenev on March 29, 2014 · 0 comments

in hematopoietic

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One of the interesting and unresolved questions in stem cell biology is how divisional history of adult stem cells correlates with their function in homeostasis. Some studies have demonstrated, that hematopoietic stem cells (HSC), once activated and divided, can come back to quiescent (dormant) state and retain function. However, this HSC homeostatic model was recently challenged by Kateri Moore’s group study, published in Stem Cell Reports.

We study the function of HSC mostly in pathological conditions, namely – transplantation model with conditioning (usually irradiation). In order to define HSC function in steady state hematopoiesis, we can not use the same “pathological models” (irradiated recipient). Two models were proposed to study the history of HSC divisions and function in homeostasis – CFSE labeling and genetic H2B method. Advantages and disadvantages of each method we discussed earlier. Markus Manz group pioneered CFSE assay for tracking of HSC divisional history and emphasized that the resolution of H2B model is too low for precise cell sorting. The recent Moore’s study was done on H2B model and claimed “good enough” resolution of HSC divisions. You can look at cell divisions gating on histogram plots from both methods and compare:

(CFSE labeling of LSK divisions after transplantation – one peak = one division, Takizawa, et al. 2011, modified. Original figure)

(GFP dilution in H2B model, representing HSC divisional kinetics, Qui et al. 2014)

I think, everybody would agree, that the biggest advantage of H2B model is in vivo labeling of dividing cells, without necessity of cell isolation and ex vivo labeling. However, we can not discount a stress-factor – doxycycline administration (it could be toxic) in order to turn GFP off and track cell divisions.

The main conclusions from Qui’s study:

Collectively, our results suggest that, during normal homeostasis, HSCs do not undergo self-renewal divisions. They also suggest that, once HSCs exit dormancy and initiate cell division, they will not return to full functional dormancy and are slated for extinction.

… we still challenge the existing dogma that HSCs self-renew to their initial functional potential once they initiate cell division under any condition. HSCs may not be immortal, nor should they be…

The authors did a giant work to characterize HSC subsets phenotypically and functionally in different states of divisional history in H2B model. Most of phenotypic studies were done on cell subsets simply subdivided on GFP-high (aka dormant = GFP4) and GFP-low (HSC with >1-2 divisions). Importantly, they found that surface phenotype correlates poorly with HSC function:

…the loss of self-renewal potential as LSK cells exit GFP4 cannot be explained by a loss of phenotypically defined LT-HSCs because the GFP3 and GFP4 fractions are almost identical, suggesting that divisional history is a better indicator of stem cell function than phenotype.

If you’re doing HSC assays (especially flow cytometry and transplantation), I’d highly recommend you to read this paper. The authors confirmed Morita’s findings for CD150 heterogeneity and provided new insight to CD34 marker for mouse HSC. The most interesting message for me was a contradiction to Kitazawa’s study and to one of my favorites – “dynamic repitition” model of HSC homeostasis (see slide 17 of presentation). I’d be very curious to get feedback from Markus Manz group about Qui’s study.

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