1. Hematopoietic stem cell heterogeneity and assay-based definitions Alexey Bersenev Journal Club Sep. 17, 2010

2. 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?

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+ HSC heterogeneity based on cell cycle kinetics

6. HSC heterogeneity based on cell cycle kinetics and metabolism 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. Use of CD150 to dissect HSC heterogeneity and lineage bias – Challen study Challen G et al. Cell Stem Cell 2010

8. 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-)

9. single cell transplant  3 subsets of LSK/CD34-/CD150 Morita Y. study - experimental setup:

10. Heterogeneity within CD150 subset of LSK/CD34- HSC CD150 1 st  2 nd transplant repopulation kinetics 1 cell transplant outcome high med neg long-term repopulation (blood chimerism at 5 months) 1 st transplant 16/40 13/40 13/40 1 st transplant lineage- bias myeloid mixed lymphoid long-term repopulation (blood chimerism at 5 months) 2 nd transplant 13/13 4/13 2/12 2 nd transplant lineage- bias myeloid mixed lymphoid

12. Functional hierarchy of pure HSCs with the same phenotype

18. 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