Assessment of Young and Aged Hematopoietic Stem Cell Activity by Competitive Serial Transplantation Assays

  • Yu Wei Zhang
  • Nina Cabezas-WallscheidEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2017)


Healthy hematopoietic stem cells (HSCs) are capable to self-renew and reconstitute the complete hematopoietic system. Upon aging, there is an increased incidence of blood-related diseases. Age-related phenotypes have been widely studied by bone marrow transplantation experiments, where reconstitution of the transplanted cells is a direct measure of HSC activity. In this protocol we describe a competitive bone marrow transplantation assay to functionally test young and old HSCs.

Key words

Hematopoietic stem cells Transplants Young Aged Bone marrow Competitive 


  1. 1.
    Weissman IL (2000) Stem cells: units of development, units of regeneration, and units in evolution. Cell 100(1):157–168PubMedCrossRefGoogle Scholar
  2. 2.
    Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132(4):631–644PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    He S, Nakada D, Morrison SJ (2009) Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol 25(1):377–406PubMedCrossRefGoogle Scholar
  4. 4.
    Seita J, Weissman IL (2010) Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med 2(6):640–653PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Cabezas-Wallscheid N, Klimmeck D, Hansson J, Lipka Daniel B, Reyes A, Wang Q, Weichenhan D, Lier A, von Paleske L, Renders S, Wünsche P, Zeisberger P, Brocks D, Gu L, Herrmann C, Haas S, Essers MAG, Brors B, Eils R, Huber W, Milsom MD, Plass C, Krijgsveld J, Trumpp A (2014) Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15(4):507–522PubMedCrossRefGoogle Scholar
  6. 6.
    Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M, Offner S, Dunant CF, Eshkind L, Bockamp E, Lió P, MacDonald HR, Trumpp A (2008) Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135(6):1118–1129PubMedCrossRefGoogle Scholar
  7. 7.
    van der Wath RC, Wilson A, Laurenti E, Trumpp A, Liò P (2009) Estimating dormant and active hematopoietic stem cell kinetics through extensive modeling of bromodeoxyuridine label-retaining cell dynamics. PLoS One 4(9):e6972PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Qiu J, Papatsenko D, Niu X, Schaniel C, Moore K (2014) Divisional history and hematopoietic stem cell function during homeostasis. Stem Cell Reports 2(4):473–490PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Bernitz JM, Kim HS, MacArthur B, Sieburg H, Moore K (2016) Hematopoietic stem cells count and remember self-renewal divisions. Cell 167(5):1296–1309.e1210PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Cantor AB, Orkin SH (2001) Hematopoietic development: a balancing act. Curr Opin Genet Dev 11(5):513–519PubMedCrossRefGoogle Scholar
  11. 11.
    Miyamoto T, Iwasaki H, Reizis B, Ye M, Graf T, Weissman IL, Akashi K (2002) Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. Dev Cell 3(1):137–147PubMedCrossRefGoogle Scholar
  12. 12.
    Adolfsson J, Månsson R, Buza-Vidas N, Hultquist A, Liuba K, Jensen CT, Bryder D, Yang L, Borge O-J, Thoren LAM, Anderson K, Sitnicka E, Sasaki Y, Sigvardsson M, Jacobsen SEW (2005) Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential: a revised road map for adult blood lineage commitment. Cell 121(2):295–306PubMedCrossRefGoogle Scholar
  13. 13.
    Forsberg EC, Serwold T, Kogan S, Weissman IL, Passegué E (2006) New evidence supporting megakaryocyte-erythrocyte potential of Flk2/Flt3+ multipotent hematopoietic progenitors. Cell 126(2):415–426PubMedCrossRefGoogle Scholar
  14. 14.
    Essers MAG, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duchosal MA, Trumpp A (2009) IFNα activates dormant haematopoietic stem cells in vivo. Nature 458:904–908CrossRefGoogle Scholar
  15. 15.
    Baldridge MT, King KY, Boles NC, Weksberg DC, Goodell MA (2010) Quiescent haematopoietic stem cells are activated by IFN-γ in response to chronic infection. Nature 465:793–797PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Takizawa H, Regoes RR, Boddupalli CS, Bonhoeffer S, Manz MG (2011) Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation. J Exp Med 208(2):273–284PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Walter D, Lier A, Geiselhart A, Thalheimer FB, Huntscha S, Sobotta MC, Moehrle B, Brocks D, Bayindir I, Kaschutnig P, Muedder K, Klein C, Jauch A, Schroeder T, Geiger H, Dick TP, Holland-Letz T, Schmezer P, Lane SW, Rieger MA, Essers MAG, Williams DA, Trumpp A, Milsom MD (2015) Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 520:549–552PubMedCrossRefGoogle Scholar
  18. 18.
    Cabezas-Wallscheid N, Buettner F, Sommerkamp P, Klimmeck D, Ladel L, Thalheimer FB, Pastor-Flores D, Roma LP, Renders S, Zeisberger P, Przybylla A, Schönberger K, Scognamiglio R, Altamura S, Florian CM, Fawaz M, Vonficht D, Tesio M, Collier P, Pavlinic D, Geiger H, Schroeder T, Benes V, Dick TP, Rieger MA, Stegle O, Trumpp A (2017) Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy. Cell 169(5):807–823.e19PubMedCrossRefGoogle Scholar
  19. 19.
    Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman RC (2004) Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood 104(8):2263–2268PubMedCrossRefGoogle Scholar
  20. 20.
    Lichtman MA, Rowe JM (2004) The relationship of patient age to the pathobiology of the clonal myeloid diseases. Semin Oncol 31(2):185–197PubMedCrossRefGoogle Scholar
  21. 21.
    Linton PJ, Dorshkind K (2004) Age-related changes in lymphocyte development and function. Nat Immunol 5:133–139PubMedCrossRefGoogle Scholar
  22. 22.
    Miller JP, Allman D (2003) The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors. J Immunol 171(5):2326–2330PubMedCrossRefGoogle Scholar
  23. 23.
    Rossi DJ, Bryder D, Zahn JM, Ahlenius H, Sonu R, Wagers AJ, Weissman IL (2005) Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 102(26):9194–9199PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Geiger H, de Haan G, Florian MC (2013) The ageing haematopoietic stem cell compartment. Nat Rev Immunol 13:376–389PubMedCrossRefGoogle Scholar
  25. 25.
    Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL (2007) Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447:725–729PubMedCrossRefGoogle Scholar
  26. 26.
    Cho RH, Sieburg HB, Muller-Sieburg CE (2008) A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells. Blood 111(12):5553–5561PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Dykstra B, Olthof S, Schreuder J, Ritsema M, de Haan G (2011) Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J Exp Med 208(13):2691–2703PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Flach J, Bakker ST, Mohrin M, Conroy PC, Pietras EM, Reynaud D, Alvarez S, Diolaiti ME, Ugarte F, Forsberg EC, Le Beau MM, Stohr BA, Méndez J, Morrison CG, Passegué E (2014) Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature 512(7513):198–202PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Tuljapurkar SR, McGuire TR, Brusnahan SK, Jackson JD, Garvin KL, Kessinger MA, Lane JT, O’Kane BJ, Sharp JG (2011) Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging. J Anat 219(5):574–581PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Ergen AV, Boles NC, Goodell MA (2012) Rantes/Ccl5 influences hematopoietic stem cell subtypes and causes myeloid skewing. Blood 119(11):2500–2509PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Harrison D (1980) Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 55(1):77–81PubMedGoogle Scholar
  32. 32.
    Shen FW, Saga Y, Litman G, Freeman G, Tung JS, Cantor H, Boyse EA (1985) Cloning of Ly-5 cDNA. Proc Natl Acad Sci U S A 82(21):7360–7363PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Yakura H, Shen FW, Bourcet E, Boyse EA (1983) On the function of Ly-5 in the regulation of antigen-driven B cell differentiation. Comparison and contrast with Lyb-2. J Exp Med 157(4):1077–1088PubMedCrossRefGoogle Scholar
  34. 34.
    Spangrude G, Heimfeld S, Weissman I (1988) Purification and characterization of mouse hematopoietic stem cells. Science 241(4861):58–62PubMedCrossRefGoogle Scholar
  35. 35.
    Waterstrat A, Liang Y, Swiderski CF, Shelton BJ, Van Zant G (2010) Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment. Blood 115(2):408–417PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Mercier FE, Sykes DB, Scadden DT (2016) Single targeted exon mutation creates a true congenic mouse for competitive hematopoietic stem cell transplantation: the C57BL/6-CD45.1 (STEM) mouse. Stem Cell Reports 6(6):985–992PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Flurkey KCJ, Harrison DE (2007) The mouse in aging research. In: Fox JG et al (eds) The mouse in biomedical research, 2nd edn. American College Laboratory Animal Medicine, Elsevier, pp 637–672CrossRefGoogle Scholar
  38. 38.
    Eaves CJ (2015) Hematopoietic stem cells: concepts, definitions, and the new reality. Blood 125(17):2605–2613PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Max Planck Institute of Immunobiology and EpigeneticsFreiburgGermany
  2. 2.International Max Planck Research School for Molecular and Cellular Biology (IMPRS-MCB)FreiburgGermany

Personalised recommendations