Spleen hsc online


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Spleen hsc online

  1. 1. Studying hematopoietic stem cells in adult spleen Alexey Bersenev Journal Club Feb. 3, 2011
  2. 2. <ul><li>Shielding of the spleen allowed mice to recover from a lethal dose of irradiation ( Jacobson LO et al., 1949 ) </li></ul><ul><li>Post-irradiated (15 min – 1h and more) spleenectomized mice survived if spleen was shielded during irradiation ( Jacobson LO, et al, 1951 ) </li></ul><ul><li>Intraperitoneal inoculation of spleen fragments from infant mice, allows to survive lethal irradiation ( Jacobson LO, et al, 1951 ) </li></ul><ul><li>HSC in mouse adult spleen probably exist, functional (radioprotective) and able to home and engraft in bone marrow </li></ul>HSC in adult spleen - background
  3. 3. <ul><li>Are HSCs in the spleen residing or circulating? </li></ul><ul><li>What markers spleen HSC express and how it different from BM HSC? </li></ul><ul><li>Can pure sorted spleen HSC rescue irradiated animal, home to bone marrow and possess multilineage engraftment? </li></ul><ul><li>How spleen and BM HSC different physiologically? (mobilization, homing signals, cell cycle, metabolism) </li></ul><ul><li>What is the niche for spleen HSC? </li></ul>HSCs in adult spleen – background Questions:
  4. 4. <ul><li>Significance: </li></ul><ul><li>For the first time spleen HSCs were isolated and studied in functional transplantation assays </li></ul><ul><li>Function of spleen HSCs was assessed on single cell level </li></ul><ul><li>For the first time cell cycle of spleen HSCs was studied </li></ul><ul><li>Residential, but not circulating nature of spleen HSCs was shown </li></ul>Study of HSC in adult spleen precisely on single cell level Morita Y, et al. Exp Hematol, Dec. 2010 online
  5. 5. Summary of transplantation assays – total BM or SP cells despite more than 10 times less frequency of HSCs in spleen, their activity is equal to BM counterparts Parameter BM SP % chimerism (1M donor+1M competitor) 50% 6.5% RU value per 1M donor cells 11.2 0.75 Repopulation pattern multilineage lymphoid-biased LTRCs frequency 1 in 30k 1 in 310k Mean activity per stem cell MAS = RU/CRU 0.34 0.39
  6. 6. Summary of in vitro and in vivo assays – sorted HSCs (LSK/CD34-) repopulating and self-renewal potentials of HSCs are similar in BM and spleen Parameter BM SP % chimerism (10 HSCs + 0.2M competitor) 16.7% 9% % chimerism in 2 nd transplant (whole pooled cells) 12.3 9.8 Single HSC transplant positive/ total 9/29 7/29 LTRC frequency roughly estimated 1 in 3 1 in 5 Repopulation pattern multilineage multilineage
  7. 7. <ul><li>Most spleen, HSCs if not all, are long-term residents </li></ul>Spleen HSCs differ from circulating HSCs Parabiosis pairs chimerism Blood Ly5.2 blood chimerism in Ly5.1 49.4% Ly5.1 blood chimerism in Ly5.2 39.2% BM SP Ly5.2+ cells in Ly5.1 mice 20.4% 44.9% Ly5.1+ cells in Ly5.2 mice 20% 32.5% Ly5.2+ HSCs in Ly5.1 mice 6.6% 8.6% Ly5.1+ HSCs in Ly5.2 mice 2.4% 9.2%
  8. 8. <ul><li>Pyronin Y staining (RNA content): </li></ul><ul><li>97.9% of G-0 HSCs in BM </li></ul><ul><li>90.9% of G-0 HSCs in SP </li></ul><ul><li>Cell cycle kinetics by BrdU labeling: </li></ul><ul><li>50% turnover time = 17 days for BM and 9 days for SP </li></ul><ul><li>mean turnover time = 25 days for BM and 13 days for SP </li></ul><ul><li>spleen HSCs cycle almost twice as often as do BM ones </li></ul>Cell cycle of spleen HSCs
  9. 9. <ul><li>HSC in adult spleen are long-term residents, not circulating </li></ul><ul><li>Spleen HSCs express the same surface as HSCs in bone marrow </li></ul><ul><li>Frequency of HSC in the spleen 10 times less than in BM (limiting dilution transplantation assay) </li></ul><ul><li>Single sorted spleen HSCs can rescue lethally irradiated mice </li></ul><ul><li>Quality of the spleen HSCs (repopulating activity, self-renewal, multilineage engraftment) the same with BM counterpart. They are functionally indentical on single cell level </li></ul><ul><li>HSCs fraction in spleen is less quiescent than in BM, Spleen HSCs cycle twice more often than BM counterpart </li></ul>Conclusions from Morita’s study:
  10. 10. <ul><li>What is the function of spleen HSCs? </li></ul><ul><li>What is the niche for spleen HSC? </li></ul><ul><li>Organ-specific intrinsic regulation – gene expression profile </li></ul><ul><li>Physiology – egression, trafficking, homing, metabolism </li></ul><ul><li>Aging </li></ul>Unresolved questions:
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