Re-thinking of hematopoietic stem cell assaysRe-thinking of hematopoietic stem cell assays

Alexey Bersenev

Journal club Aug 14, 2009Journal 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

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

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 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

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

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

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:

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

Leu

kem

ia

Str

ess

senescencesenescence

exhaustion apoptosisapoptosisquiescence & self-renewal

stress (serial BMT; myelosupression)

quiescence & self-renewal

leukemia leukemia progressionprogression

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

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

Part 5

mechanisms

HSC

proliferation (cell cycle entry)

MPDMPD

myeloid progenitors

quiescence & self-renewal

LSC

mutation

LSC LSC

aberrant clone

JunB-deficient mice:

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:

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:

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

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:

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