Revised: Cell cycle kinetics of hematopoietic stem cells

Alexey Bersenev

Journal Club May 12, 2011

Definitions

1. Quiescence – reversible cell cycle arrest (exit); G-0 phase of cell cycle

2. Dormancy – metabolically low (hibernating) state of quiescent cells. Not well defined. Frequently used as a synonym of quiescence

Cell cycle kinetics:

turnover rate –

one division per ... days

# of divisions during lifetime

Cycling cell fate decisions:

quiescencesenescence

apoptosis

proliferation

self-renewal differentiation/ maturation

dormancy?

Current general assumptions from HSC biology:

1. True HSC with robust self-renewing and repopulating capacity are quiescent (non-dividing or very slow dividing)

2. The dormancy model of the somatic stem cell population implies the co-existence of phenotypically similar – “deeply quiescent” (dormant) and active stem cells

activedormant

feedback loop

long-termprogenitors

short-term

HSC

quiescence

self-renewal

Conventional model

Dormancy model

mat

ure

bloo

d ce

lls

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

Dormancy model of hematopoiesis:

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 +++ +

Dormant versus active HSC

Andreas Trumpp 2008-2009, Linheng Li, Hans Clevers 2010

Unresolved questions from dormancy model:

1. How small fraction of slow or non-dividing dormant HSC can maintain rapid blood turnover for entire life in steady state conditions? Their contribution in steady state hematopoiesis was not assess properly.

2. How dormant HSC contribute to rescuing of hematopoiesis in emergency – by differentiation or self-renewal division?

3. What is the link between divisional history and function of HSC?

Studying of stem cell quiescence - methodology

1. DNA versus RNA content by flow cytometry allows to separate G-0 and G-1 phases of cell cycle (Hoechest and Pyronin Y)

2. DNA label-retaining assays (BrdU)

3. Histon-2B (H2B) label-retaining assay in vivo

4. CFSE label-retaining assay

BrdU method

1. Reproducible

1. Done on dead (fixed) cells

2. Presence of BrdU (or other pyrimidine analogs) induces cells to cycle due to some toxicity

3. BrdU labeling doesn’t allow clear separation of slow dividing stem cells versus non-dividing (G-0 versus G-1)

4. Does not label all HSC (missing some quiescent ones)

advantages:

disadvantages:

H2B method

1. Genetic labeling! (don’t need ex vivo step for labeling)

2. Allows to study quiescent HSC in vivo (cell sorting and transplant)

3. Does not impair HSC function

4. Reproducible (since 2008 used in more than 30 labs)

1. Does not allow to study HSC function based on divisional history due to low resolution

advantages:

disadvantages:

CFSE method –Proposed for studying HSC cell cycle kinetics by Takizawa H, from Manz group, 2011

1. Studying quiescence in vivo

2. Tracking divisional history of HSC

3. Does not impair HSC function

4. Very easy and rapid labeling (7 minutes)

1. Need to perform transplantation assays

2. Unknown reproducibility due novelty for stem cell biology

advantages:

disadvantages:

Significance of Takizawa’s study:

1. Precisely assess contribution of dividing and non-dividing HSC in steady state hematopoiesis

2. Challenges the notion that stem cell function (self-renewal and multilineage reconstitution) directly associated with quiescence

3. Challenges the use of BrdU for proper quiescence assessment

4. Points to indications when and how HSC switch from actively cycling state to quiescence

Conclusions (methodology):

1. CFSE labeling is a good assessment of stem cell quiescence, allows high resolution divisional history and could complement H2B method

2. Low divisional resolution of BrdU retention, therefore use of BrdU for assessment of HSC quiescence is not accurate

3. Steady state hematopoiesis should be studied in nonirradiated recipients

Conclusions (HSC biology):

1. Life-long steady state hematopoiesis maintained equally by frequently cycling and quiescent HSC

2. Progressive divisional history does not lead to immediate loss of HSC function (exhaustion)

3. Cycling HSC with extensive proliferative history can slow down and go into quiescence retaining functional potential

Challenge the dormancy model:

Long-term repopulation HSC are not necessarily permanently split into subpopulations with different cycling kinetics

Hypothetical models of steady state hematopoiesis

turnover rate:

HSC divide every 17.8 days (BrdU/ biotin)

aHSC divide every 9-36 days(H2B)

dHSC divide every 56-145d

HSC divide every 39 days –18 divisions during lifetime

(CFSE)

“Dynamic repetition” model -

Some HSC dominate blood formation for a time, subsequently enter a quiescent state in which other HSC increase hematopoietic contribution, and get reactivated again – repetitive cycles

“dynamic equilibrium” between quiescent fraction and cycling fraction within the HSC population

For the first time was proposed by Ingmar Glauche et al. Stem cell proliferation and quiescence--two sides of the same coin. PLoS Comput Biol. 2009 Jul;5(7):e1000447

2009 Glauche concept:

HSC flexibly and reversibly adapt their cycling state according to systemic needs

Unresolved questions:

1. Why two sophisticated methods gave us a different answer about the link between divisional dynamics and HSC function?

2. How to sync H2B and CSFE methods?

3. How HSC surface markers correlate with quiescence reversibility?