Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Re-thinking of hematopoietic stem cell assays Alexey Bersenev Journal club Aug 14, 2009
What assay do you choose when designing your project? <ul><li>A. The one that is considered right up-to-date in the field ...
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 1994 Nat Med 1997 Human AML L...
Example 2 Reya T. lab Nature 2009 Hedgehog pathway is essential  for normal HSC function and leukemic stem cells Model :  ...
Studying the same scientific problem by using different assays and models could lead to completely different results  and ...
<ul><li>Short-term assays: </li></ul><ul><li>In vitro: </li></ul><ul><li>Immunophenotyping of HSC/progenitors (flow cytome...
<ul><li>Competing cells  (whole BM vs. progenitors (c-Kit+ or Sca-1+) depleted, cell number) </li></ul><ul><li>Donor (test...
<ul><li>Significance : </li></ul><ul><li>Valuable lesson on the interpretation of classical stem cell biology assays in mu...
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 v...
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 fu...
Quiescent fraction of  BM hematopoietic stem/progenitor cells could be considered as an equivalent of LT-HSC  Part 2 Quies...
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-/CD...
HSC LSC Leukemia Stress senescence exhaustion apoptosis quiescence & self-renewal stress   (serial BMT; myelosupression) q...
Part 3 Link between quiescence and function of HSCs Cell cycle activation/entry (loss of quiescence) of HSC associated wit...
250 WT LSK/Flk2-/CD150+/CD48- 56% chimerism 250 WT LSK/Flk2-/G0+ 25% chimerism 250 JunB LSK/Flk2-/CD150+/CD48- 82% chimeri...
Challenge quiescence per se is not a defining characteristic of LT-HSCs and should not be used as the sole criterion for i...
Mutations or stress causing increased cell cycle rate typically result in stem cell exhaustion Part 4 Link between quiesce...
HSC HSC JunB deficient Normal HSC HSC exhaustion and  death quiescence & self-renewal stress   (5-FU BM cycling cell ablat...
Part 5 mechanisms
HSC proliferation (cell cycle entry) MPD myeloid progenitors quiescence & self-renewal LSC mutation LSC LSC aberrant clone...
<ul><li>transplant a highly purified cell population when assessing LT-HSC numbers and properties in situations in which a...
Q: Should be reevaluate findings from previous reports in which HSC functions were characterized using nonpurified cell po...
Other possible reasons of variability in HSC assays: <ul><li>Cell to cell variability </li></ul><ul><li>Mouse to mouse var...
Valid and commonly used model Irradiated   vs.  nonablated   host: Valid but not widely used Virtually all stem cell engra...
Literature: <ul><li>Purton LE, Scadden D.  Limiting factors in murine hematopoietic stem cell assays.   Cell Stem Cell 200...
Upcoming SlideShare
Loading in …5
×

Jc Rethinking Of Hsc Assays

2,026 views

Published on

Published in: Health & Medicine, Technology
  • Be the first to comment

  • Be the first to like this

Jc Rethinking Of Hsc Assays

  1. 1. Re-thinking of hematopoietic stem cell assays Alexey Bersenev Journal club Aug 14, 2009
  2. 2. What assay do you choose when designing your project? <ul><li>A. The one that is considered right up-to-date in the field </li></ul><ul><li>I usually set it up from the scratch </li></ul><ul><li>Whatever we have available in the lab/ building/ campus </li></ul><ul><li>I don’t care, whatever my PI told me </li></ul><ul><li>All of the above </li></ul>test: At the end none of them could be right
  3. 3. Stem cell research right now is everything about assays and models Why is it important?
  4. 4. Example 1 Human AML LSC phenotype : Only Lin-/CD34+/CD38– can give transplantable AML Nature 1994 Nat 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
  5. 5. Example 2 Reya T. lab Nature 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. labs Cell Stem Cell 2009 Hedgehog pathway is dispensable for normal HSC function and leukemia
  6. 6. 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?
  7. 7. <ul><li>Short-term assays: </li></ul><ul><li>In vitro: </li></ul><ul><li>Immunophenotyping of HSC/progenitors (flow cytometry) </li></ul><ul><li>Colony-forming assay (CFC) </li></ul><ul><li>In vivo: </li></ul><ul><li>Colony-forming unit-spleen (CFU-S) </li></ul>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
  8. 8. <ul><li>Competing cells (whole BM vs. progenitors (c-Kit+ or Sca-1+) depleted, cell number) </li></ul><ul><li>Donor (test) cells (whole BM vs purified progenitors or HSC populations; freshly isolated vs cultured HSC ) </li></ul><ul><li>Donor (test) cells from single mouse or pulled from few mice </li></ul><ul><li>Donor cell % and lineages used to determine the number of negative/positive mice (1% vs 0.1%; multilineage vs any 1 lineage) </li></ul><ul><li>Time posttransplant at which the results are analyzed (3 or 6 months) </li></ul><ul><li>Method of detecting donor vs host cells (CD45.1-CD45.2; Y-chromosome, GFP) </li></ul><ul><li>Recipient conditioning (lethal vs. non-irradiated vs. sublethal) </li></ul>Some questions about the “gold standard” under discussion:
  9. 9. <ul><li>Significance : </li></ul><ul><li>Valuable lesson on the interpretation of classical stem cell biology assays in mutant models </li></ul><ul><li>New insights into the role of JunB in HSC biology and early MPD development </li></ul><ul><li>Possible impact : </li></ul><ul><li>re-evaluation of HSC assays for HSC function in leukemia models </li></ul>
  10. 10. 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
  11. 11. Jordan CT and Guzman M Cancer Cell 2009
  12. 12. Using whole BM cells for competitive repopulation assays is potentially unreliable method of determining HSC number and function in pathological situations Challenge: Conclusion:
  13. 13. 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:
  14. 14. 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
  15. 15. HSC LSC Leukemia Stress senescence exhaustion apoptosis quiescence & self-renewal stress (serial BMT; myelosupression) quiescence & self-renewal leukemia progression virtually unexhausted model
  16. 16. 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:
  17. 17. 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!
  18. 18. 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:
  19. 19. 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:
  20. 20. HSC HSC JunB deficient Normal HSC 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
  21. 21. Part 5 mechanisms
  22. 22. HSC proliferation (cell cycle entry) MPD myeloid progenitors quiescence & self-renewal LSC mutation LSC LSC aberrant clone JunB-deficient mice:
  23. 23. <ul><li>transplant a highly purified cell population when assessing LT-HSC numbers and properties in situations in which artificially introduced genetic alterations </li></ul><ul><li>isolating HSPC based on G-0 (quiescent) phase of cell cycle could not be used to assess HSC function </li></ul><ul><li>side by side confirmation of results from whole BM transplant to pure HSC populations </li></ul><ul><li>side by side confirmation of findings on cell cycle studying from LSK to LSK/CD34- to LSK/SLAM </li></ul>Lessons to learn:
  24. 24. 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
  25. 25. Other possible reasons of variability in HSC assays: <ul><li>Cell to cell variability </li></ul><ul><li>Mouse to mouse variability </li></ul><ul><li>Stochastic variability </li></ul><ul><li>Circadian rhythm </li></ul><ul><li>Nature of engrafted host – irradiated vs. nonablated </li></ul><ul><li>Continuum cell-cycle-related phenotype fluctuations </li></ul>Quesenberry and Dooner. Exp Hematol 2009
  26. 26. 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
  27. 27. Literature: <ul><li>Purton LE, Scadden D. Limiting factors in murine hematopoietic stem cell assays. Cell Stem Cell 2007;1:263 </li></ul><ul><li>Quesenberry PJ, Dooner GJ, Dooner MS. Problems in the promised land: Status of adult marrow stem cell biology. Exp Hematol 2009;37:775 </li></ul><ul><li>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 </li></ul>

×