Jc Rethinking Of Hsc Assays

Re-thinking of hematopoietic stem cell assays Alexey Bersenev Journal club Aug 14, 2009

What assay do you choose when designing your project?
A. The one that is considered right up-to-date in the field
I usually set it up from the scratch
Whatever we have available in the lab/ building/ campus
I don’t care, whatever my PI told me
All of the above
test: At the end none of them could be right

Stem cell research right now is everything about assays and models Why is it important?

Example 1 Human AML LSC phenotype : Only Lin-/CD34+/CD38– can give transplantable AML Nature 1994 Nat Med 1997 Human AML LSC phenotype : Lin-/CD34+/CD38 - and Lin-/CD34+/CD38+ both equally can give transplantable AML Proof of cancer stem cell concept for AML disproof of cancer stem cell concept for AML? Model : NOD/SCID mice Model : NOG mice Blood 2008 Identification of human leukemic stem cells and cancer stem cell concept

Example 2 Reya T. lab Nature 2009 Hedgehog pathway is essential for normal HSC function and leukemic stem cells Model : Vav-Cre mice Model : Mx1-Cre mice Hedgehog signaling pathway in HSC and leukemia Gilliland G. and Aifantis A. labs Cell Stem Cell 2009 Hedgehog pathway is dispensable for normal HSC function and leukemia

Studying the same scientific problem by using different assays and models could lead to completely different results and eventually mislead medical the community in clinical trials So… Where is a truth?

Short-term assays:
In vitro:
Immunophenotyping of HSC/progenitors (flow cytometry)
Colony-forming assay (CFC)
In vivo:
Colony-forming unit-spleen (CFU-S)
Brief summary of HSC assays In vivo: Competitive repopulation (Harrison, 1980) Limiting dilution Serial transplant Long-term assays: In vitro: Cobblestone area-forming cells (CAFC) or long-term culture-initiating cells (LTC-IC) GOLD STANDARD Purton and Scadden Cell Stem Cell 2007

Competing cells (whole BM vs. progenitors (c-Kit+ or Sca-1+) depleted, cell number)
Donor (test) cells (whole BM vs purified progenitors or HSC populations; freshly isolated vs cultured HSC )
Donor (test) cells from single mouse or pulled from few mice
Donor cell % and lineages used to determine the number of negative/positive mice (1% vs 0.1%; multilineage vs any 1 lineage)
Time posttransplant at which the results are analyzed (3 or 6 months)
Method of detecting donor vs host cells (CD45.1-CD45.2; Y-chromosome, GFP)
Recipient conditioning (lethal vs. non-irradiated vs. sublethal)
Some questions about the “gold standard” under discussion:

Significance :
Valuable lesson on the interpretation of classical stem cell biology assays in mutant models
New insights into the role of JunB in HSC biology and early MPD development
Possible impact :
re-evaluation of HSC assays for HSC function in leukemia models

The best source of donor cells in HSC studies of adult mutant or non-steady-state mice is whole BM cells Part 1 whole BM vs. pure HSC transplantation in competitive repopulation assay general assumption: Purton and Scadden Cell Stem Cell 2007

Jordan CT and Guzman M Cancer Cell 2009

Using whole BM cells for competitive repopulation assays is potentially unreliable method of determining HSC number and function in pathological situations Challenge: Conclusion:

Quiescent fraction of BM hematopoietic stem/progenitor cells could be considered as an equivalent of LT-HSC Part 2 Quiescence and cell cycle kinetics HSC could be isolated based on G-0 cell cycle phase used in BMT assays Assumption:

WT LSK/Flk2- 76% G-0 cells WT LSK/Flk2-/CD150+/CD48- 96% G-0 cells WT LSK/Flk2-/CD150+/CD48+ 55% G-0 cells WT LSK/Flk2-/CD34- 95% G-0 cells WT LSK/Flk2-/CD34+ 55% G-0 cells JunB LSK/Flk2-/CD150+/CD48- 98% G-0 cells WT LSK/Flk2 + 47% G-0 cells JunB LSK/Flk2-/CD34- 52% G-0 cells

HSC LSC Leukemia Stress senescence exhaustion apoptosis quiescence & self-renewal stress (serial BMT; myelosupression) quiescence & self-renewal leukemia progression virtually unexhausted model

Part 3 Link between quiescence and function of HSCs Cell cycle activation/entry (loss of quiescence) of HSC associated with loss of their function Dogma:

250 WT LSK/Flk2-/CD150+/CD48- 56% chimerism 250 WT LSK/Flk2-/G0+ 25% chimerism 250 JunB LSK/Flk2-/CD150+/CD48- 82% chimerism Engraftment: 250 JunB LSK/Flk2-/G0+ 0% chimerism No link between quiescence and engraftment!

Challenge quiescence per se is not a defining characteristic of LT-HSCs and should not be used as the sole criterion for identifying this cell population Conclusion:

Mutations or stress causing increased cell cycle rate typically result in stem cell exhaustion Part 4 Link between quiescence and HSC function (proliferation and exhaustion) Dogma:

HSC HSC JunB deficient Normal HSC HSC exhaustion and death quiescence & self-renewal stress (5-FU BM cycling cell ablation) proliferation (cell cycle entry) progenitors Same exhaustion, but increased cell cycle rate

Part 5 mechanisms

HSC proliferation (cell cycle entry) MPD myeloid progenitors quiescence & self-renewal LSC mutation LSC LSC aberrant clone JunB-deficient mice:

transplant a highly purified cell population when assessing LT-HSC numbers and properties in situations in which artificially introduced genetic alterations
isolating HSPC based on G-0 (quiescent) phase of cell cycle could not be used to assess HSC function
side by side confirmation of results from whole BM transplant to pure HSC populations
side by side confirmation of findings on cell cycle studying from LSK to LSK/CD34- to LSK/SLAM
Lessons to learn:

Q: Should be reevaluate findings from previous reports in which HSC functions were characterized using nonpurified cell populations? Discussion: Immunophenotypical methods to isolate HSCs for competitive repopulation assays may not provide accurate results when assessing HSC content in mutant mice models (potential discrepancy between phenotype and function) Purton and Scadden Cell Stem Cell 2007 Loss of HSC with purification in nonablated recipient model. Studies on unseperated BM more biologically relevant Quesenberry and Dooner. Exp Hematol 2009

Other possible reasons of variability in HSC assays:
Cell to cell variability
Mouse to mouse variability
Stochastic variability
Circadian rhythm
Nature of engrafted host – irradiated vs. nonablated
Continuum cell-cycle-related phenotype fluctuations
Quesenberry and Dooner. Exp Hematol 2009

Valid and commonly used model Irradiated vs. nonablated host: Valid but not widely used Virtually all stem cell engrafted Doesn’t measure level of engraftment at the stem cell level Monitor all of engraftable stem cells Homing and engraftement of HSC lower Homing and engraftement of HSC higher Engraftment in irreversibly damaged and toxic BM environment Engraftment in natural BM environment No competition for BM niches Competition for BM niches occupancy Quesenberry and Dooner. Exp Hematol 2009

Literature:
Purton LE, Scadden D. Limiting factors in murine hematopoietic stem cell assays. Cell Stem Cell 2007;1:263
Quesenberry PJ, Dooner GJ, Dooner MS. Problems in the promised land: Status of adult marrow stem cell biology. Exp Hematol 2009;37:775
Guzman M, Jordan CT. Lessons learned from the study of JunB: New insights for normal and leukemia stem cell biology . Cancer Cell 2009; 15:252

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Jc Rethinking Of Hsc Assays

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