HSC_heterogeneity_definition

Hematopoietic stem cell heterogeneityHematopoietic stem cell heterogeneityandand

assay-based definitionsassay-based definitions

Alexey Bersenev

Journal Club Sep. 17, 2010

The problem of Hematopoietic Stem Cell (HSC) heterogeneity:

Studies (n~10) demonstrate that even if we can prospectively isolate highly pure HSC based on marker expression (for example: LSK/CD34-/Flk2-/CD48-/CD150+),

They are retaining a high degree of heterogeneity on the functional level

Could HSC be properly assessed on single cell level or do we always have to deal with population?

Functional heterogeneity in highly pure HSC:

1. Engraftment kinetics

2. Lineages skewing

3. Self-renewal capacity

4. Phase of cell cycle

5. Metabolism

HSC heterogeneity assessed in single cell transplant

1. Four different patterns of reconstitution after single sorted HSCs (CD45mid/Lin-/ Rho−/SP) Dykstra et. al. Cell Stem Cell 2007

2. Myeloid-biased and lymphoid-biased HSCs can be prospectively isolated (SP/CD150) Challen G et al. Cell Stem Cell 2010

3. Identification of distinct HSCs based on difference in CD150 expression (LSK/CD34-/CD48-/CD150+) Morita Y et al. JEM 2010

activedormant

feedback loop

long-term

progenitors

short-term

HSC

quiescence

self-renewal

Conventional model

Dormancy model

mat

ure

blo

od

cel

ls

LSK/34-/Flk2-/48-/150+

HSC heterogeneity based on cell cycle kinetics

characteristics dormant HSC active HSCsynonyms quiescent primed, self-renewing

phenotype LSK/CD34-/Flk2-/CD48-/CD150+

% in HSC pool ~ 15% ~ 85%

quiescence +++ +

self-renewal rate + +++

proliferation + +++

replication machinery off on

metabolism hibernation/hypoxia active

function repair in emergency normal blood turnover

niche endosteal vascular

signaling Wnt-off/ BMP-on Wnt-on/ BMP-off

activation signals active HSC depletion/ INF-α progenitors delpetion

feedback loop +++ +

HSC heterogeneity based on cell cycle kinetics and metabolism

Use of CD150 to dissect HSC heterogeneity

and lineage bias – Challen study

Challen G et al. Cell Stem Cell 2010

Use of CD150 to dissect HSC heterogeneity – Kent - Eaves study

Kent D et al. Blood 2009

Differentiation expression of CD150 on EPCR+/CD48-/CD45+ HSC dissects subsets with durable self-renewal capacity (CD150+) and limited self-renewal (CD150-)

single cell transplant 3 subsets of LSK/CD34-/CD150

Morita Y. study - experimental setup:

Heterogeneity within CD150 subset of LSK/CD34- HSC

1 cell transplant outcome high med neglong-term repopulation

(blood chimerism at 5 months) 1st transplant

16/40 13/40 13/40

1st transplant lineage- bias myeloid mixed lymphoid

long-term repopulation (blood chimerism

at 5 months) 2nd transplant13/13 4/13 2/12

2nd transplant lineage- bias myeloid mixed lymphoid

CD150

1st 2nd transplant repopulation kinetics

Heterogeneity within CD150-high subset:

Latent CD150-high:

1. 1 out of 10 CD150-high

2. Late onset of repopulation (after 4 month) at primary transplant (some mice engrafted only by 8 months after transplant)

3. Not always multilineage reconstitution at 1st transplant, myeloid-biased

4. Could be very low level of engraftment at 1st transplant (0.3% is a threshold for positively reconstituted mouse, not 1%)

5. Rapid and significant multilineage engraftment at 2nd transplant = enhanced self-renewal capability

Other 2 types of CD150-high:

• With balanced reconstitution at 1st and 2nd transplants (no bias)

• Myeloid-biased with high repopulation without engraftment delay

Functional hierarchy of pure HSCs with the same phenotype

Conclusions from Morita study:

1. Functionally distinct long-term repopulating HSC can be identified based on level of CD150 expression

2. HSCs with high expression of CD150 and latent repopulation coupled with robust self-renewal potential represent the top hierarchy in mouse hematopoiesis

3. Taking in account high degree of functional heterogeneity, assay-based definition of HSC should be modify

Current assay-based definition of HSC:

1. multilineage (myelo-, T- and B-lymphoid) reconstitution

2. assessed by blood chimerism

3. with donor’s contribution to each lineage 1% or more

4. at primary transplant

5. at 4 months

Challenges of current definition from Morita study:

1. For some HSC threshold of positive reconstitution 0.3% and below (sometimes=0) at 1st transplant

2. Some HSCs have delayed engraftment up to 6-8 months at 1st transplant

3. Picture of reconstitution can completely change at 2nd transplant

4. Blood chimerism can under- or overestimate HSC engraftment and sometimes does not correlate with bone marrow engraftment

Revised assay-based HSC definition:

1. multilineage (myelo-, T- and B-lymphoid) reconstitution in the blood and bone marrow

2. assessed at least after 2nd transplant from all primary mice

3. at 4-6 month after transplant

HSC can not be assessed after primary transplant!

Serial transplant is a must!

Questions:

1. Time of reconstitution assessment after primary transplant – 4-6-8 months?

2. Wait longer than 4 months or do 2nd transplant right away for all (“positive and negative”) recipients and assess HSC only after 2nd ?

3. What is the threshold for lineage “positive reconstitution” after 2nd transplant?

4. Should we use erythroid lineage markers and assess marrow?

5. How to qualify the following patterns of reconstitution:Gr1+ or low/Mac1-/CD19+/CD(4+8)+

Gr1+/Mac1+/CD19+/CD(4+8)-Gr1+/Mac1+/CD19-/CD(4+8)+

Downside of revised definition:

“However, this definition requires expensive and time-consuming experiments. Therefore, novel assays that permit rapid and efficient detection of all sorts of HSCs need to be developed.”

Morita Y et al. JEM 2010

Conclusions from HSC heterogeneity studies:

1. Highly pure HSC possess functional heterogeneity on single cell level. Intrinsic lineage bias was demonstrated. Influence of extrinsic factors still unknown.

2. Assessment of HSC as population could be more accurate than single cell. Back to whole bone marrow transplant?

3. Taking in account high degree of functional heterogeneity, assay-based definition of HSC should be modify

HSC heterogeneity - references:

1. Muller-Sieburg et al. Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness. Blood 2004

2. Dykstra B. et al. Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell 2007

3. Challen G. et al. Myeloid-biased and lymphoid-biased HSCs can be prospectively isolated (SP/CD150). Cell Stem Cell 2010

4. Kent D. et al. Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood 2009

5. Morita Y. et al. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. JEM 2010

6. Wilson A. et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 2008

7. Benveniste P. et al. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell 2010

8. Beerman I. et al. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. PNAS 2010