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Hsc quiescence online


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Hsc quiescence online

  1. 1. Revised: Cell cycle kinetics of hematopoietic stem cells Alexey Bersenev Journal Club May 12, 2011
  2. 2. Definitions <ul><li>Quiescence – reversible cell cycle arrest (exit); G-0 phase of cell cycle </li></ul><ul><li>Dormancy – metabolically low (hibernating) state of quiescent cells. Not well defined. Frequently used as a synonym of quiescence </li></ul>Cell cycle kinetics: turnover rate – one division per ... days # of divisions during lifetime
  3. 3. Cycling cell fate decisions: quiescence senescence apoptosis proliferation self-renewal differentiation/ maturation dormancy?
  4. 4. Current general assumptions from HSC biology: <ul><li>True HSC with robust self-renewing and repopulating capacity are quiescent (non-dividing or very slow dividing) </li></ul><ul><li>The dormancy model of the somatic stem cell population implies the co-existence of phenotypically similar – “deeply quiescent” (dormant) and active stem cells </li></ul>
  5. 5. active dormant feedback loop long-term progenitors short-term HSC quiescence self-renewal Conventional model Dormancy model mature blood cells LSK/34-/Flk2-/48-/150+ Dormancy model of hematopoiesis:
  6. 6. Dormant versus active HSC Andreas Trumpp 2008-2009, Linheng Li, Hans Clevers 2010 characteristics dormant HSC active HSC synonyms 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 +++ +
  7. 7. Unresolved questions from dormancy model: <ul><li>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. </li></ul><ul><li>How dormant HSC contribute to rescuing of hematopoiesis in emergency – by differentiation or self-renewal division? </li></ul><ul><li>What is the link between divisional history and function of HSC? </li></ul>
  8. 8. Studying of stem cell quiescence - methodology <ul><li>DNA versus RNA content by flow cytometry allows to separate G-0 and G-1 phases of cell cycle (Hoechest and Pyronin Y) </li></ul><ul><li>DNA label-retaining assays (BrdU) </li></ul><ul><li>Histon-2B (H2B) label-retaining assay in vivo </li></ul><ul><li>CFSE label-retaining assay </li></ul>
  9. 9. BrdU method <ul><li>Reproducible </li></ul><ul><li>Done on dead (fixed) cells </li></ul><ul><li>Presence of BrdU (or other pyrimidine analogs) induces cells to cycle due to some toxicity </li></ul><ul><li>BrdU labeling doesn’t allow clear separation of slow dividing stem cells versus non-dividing (G-0 versus G-1) </li></ul><ul><li>Does not label all HSC (missing some quiescent ones) </li></ul>advantages: disadvantages:
  10. 10. H2B method <ul><li>Genetic labeling! (don’t need ex vivo step for labeling) </li></ul><ul><li>Allows to study quiescent HSC in vivo (cell sorting and transplant) </li></ul><ul><li>Does not impair HSC function </li></ul><ul><li>Reproducible (since 2008 used in more than 30 labs) </li></ul><ul><li>Does not allow to study HSC function based on divisional history due to low resolution </li></ul>advantages: disadvantages:
  11. 11. CFSE method – Proposed for studying HSC cell cycle kinetics by Takizawa H, from Manz group, 2011 <ul><li>Studying quiescence in vivo </li></ul><ul><li>Tracking divisional history of HSC </li></ul><ul><li>Does not impair HSC function </li></ul><ul><li>Very easy and rapid labeling (7 minutes) </li></ul><ul><li>Need to perform transplantation assays </li></ul><ul><li>Unknown reproducibility due novelty for stem cell biology </li></ul>advantages: disadvantages:
  12. 12. Significance of Takizawa’s study : <ul><li>Precisely assess contribution of dividing and non-dividing HSC in steady state hematopoiesis </li></ul><ul><li>Challenges the notion that stem cell function (self-renewal and multilineage reconstitution) directly associated with quiescence </li></ul><ul><li>Challenges the use of BrdU for proper quiescence assessment </li></ul><ul><li>Points to indications when and how HSC switch from actively cycling state to quiescence </li></ul>
  13. 13. Conclusions (methodology): <ul><li>CFSE labeling is a good assessment of stem cell quiescence, allows high resolution divisional history and could complement H2B method </li></ul><ul><li>Low divisional resolution of BrdU retention, therefore use of BrdU for assessment of HSC quiescence is not accurate </li></ul><ul><li>Steady state hematopoiesis should be studied in nonirradiated recipients </li></ul>
  14. 14. Conclusions (HSC biology): <ul><li>Life-long steady state hematopoiesis maintained equally by frequently cycling and quiescent HSC </li></ul><ul><li>Progressive divisional history does not lead to immediate loss of HSC function (exhaustion) </li></ul><ul><li>Cycling HSC with extensive proliferative history can slow down and go into quiescence retaining functional potential </li></ul>
  15. 15. Challenge the dormancy model: Long-term repopulation HSC are not necessarily permanently split into subpopulations with different cycling kinetics
  16. 16. 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)
  17. 17. “ 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
  18. 18. Unresolved questions: <ul><li>Why two sophisticated methods gave us a different answer about the link between divisional dynamics and HSC function? </li></ul><ul><li>How to sync H2B and CSFE methods? </li></ul><ul><li>How HSC surface markers correlate with quiescence reversibility? </li></ul>