1. Re-thinking of hematopoietic stem cell assays Alexey Bersenev Journal club Aug 14, 2009

3. Stem cell research right now is everything about assays and models Why is it important?

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. 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. 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?

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. Jordan CT and Guzman M Cancer Cell 2009

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. 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. 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. HSC LSC Leukemia Stress senescence exhaustion apoptosis quiescence & self-renewal stress (serial BMT; myelosupression) quiescence & self-renewal leukemia progression virtually unexhausted model

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. 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. 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. 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. 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. Part 5 mechanisms

22. HSC proliferation (cell cycle entry) MPD myeloid progenitors quiescence & self-renewal LSC mutation LSC LSC aberrant clone JunB-deficient mice:

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

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