Jc Rethinking Of Hsc Assays
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Re-thinking of hematopoietic stem cell assaysRe-thinking of hematopoietic stem cell assays
Alexey Bersenev
Journal club Aug 14, 2009Journal club Aug 14, 2009
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What assay do you choose when designing your project?
A. The one that is considered right up-to-date in the field
B. I usually set it up from the scratch
C. Whatever we have available in the lab/ building/ campus
D. I don’t care, whatever my PI told me
E. All of the above
test:
At the end none of them could be right
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Stem cell research right now is everything about assays and models
Why is it important?
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Example 1
Human AML LSC phenotype:Only Lin-/CD34+/CD38– can give
transplantable AML
Nature 1994Nat 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
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Example 2
Reya T. labNature 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. labsCell Stem Cell 2009
Hedgehog pathway is dispensable for normal HSC
function and leukemia
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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?
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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
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1. Competing cells (whole BM vs. progenitors (c-Kit+ or Sca-1+) depleted, cell number)
2. Donor (test) cells (whole BM vs purified progenitors or HSC populations; freshly isolated vs cultured HSC )
3. Donor (test) cells from single mouse or pulled from few mice
4. Donor cell % and lineages used to determine the number of negative/positive mice (1% vs 0.1%; multilineage vs any 1 lineage)
5. Time posttransplant at which the results are analyzed (3 or 6 months)
6. Method of detecting donor vs host cells (CD45.1-CD45.2; Y-chromosome, GFP)
7. Recipient conditioning (lethal vs. non-irradiated vs. sublethal)
Some questions about the “gold standard” under discussion:
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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
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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
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Jordan CT and Guzman M Cancer Cell 2009
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Using whole BM cells for competitive repopulation assays is potentially unreliable method of determining HSC number and function in pathological situations
Challenge:
Conclusion:
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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:
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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
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HSC
LSC
Leu
kem
ia
Str
ess
senescencesenescence
exhaustion apoptosisapoptosisquiescence & self-renewal
stress (serial BMT; myelosupression)
quiescence & self-renewal
leukemia leukemia progressionprogression
virtually unexhausted
model
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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:
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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!
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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:
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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:
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HSC
HSC
Jun
B d
efic
ien
tN
orm
al
HS
C
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
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Part 5
mechanisms
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HSC
proliferation (cell cycle entry)
MPDMPD
myeloid progenitors
quiescence & self-renewal
LSC
mutation
LSC LSC
aberrant clone
JunB-deficient mice:
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1. transplant a highly purified cell population when assessing LT-HSC numbers and properties in situations in which artificially introduced genetic alterations
2. isolating HSPC based on G-0 (quiescent) phase of cell cycle could not be used to assess HSC function
3. side by side confirmation of results from whole BM transplant to pure HSC populations
4. side by side confirmation of findings on cell cycle studying from LSK to LSK/CD34- to LSK/SLAM
Lessons to learn:
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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
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Other possible reasons of variability in HSC assays:
1. Cell to cell variability
2. Mouse to mouse variability
3. Stochastic variability
4. Circadian rhythm
5. Nature of engrafted host – irradiated vs. nonablated
6. Continuum cell-cycle-related phenotype fluctuations
Quesenberry and Dooner. Exp Hematol 2009
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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
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Literature:
1. Purton LE, Scadden D. Limiting factors in murine hematopoietic stem cell assays. Cell Stem Cell 2007;1:263
2. Quesenberry PJ, Dooner GJ, Dooner MS. Problems in the promised land: Status of adult marrow stem cell biology. Exp Hematol 2009;37:775
3. 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