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Ex Vivo Assays to Study Self-Renewal, Long-Term Expansion, and Leukemic Transformation of Genetically Modified Human Hematopoietic and Patient-Derived Leukemic Stem Cells

  • Pallavi Sontakke
  • Marco Carretta
  • Marta Capala
  • Hein Schepers
  • Jan Jacob SchuringaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1185)

Abstract

With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34+ cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.

Key words

Leukemic stem cell (LSC) Acute myeloid leukemia (AML) Leukemic stem cell self-renewal Ex vivo assay Bone marrow stromal coculture Lentiviral transduction of primary patient cells 

Notes

Acknowledgements

We would like to acknowledge all members of the Experimental Hematology lab for helpful discussions. This work was supported by grants from the NWO (VENI 91611105, VIDI 91796312), KWF (2009-4411), and EU (FP7 EuroCSC ITN).

References

  1. 1.
    Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 6464:645–648CrossRefGoogle Scholar
  2. 2.
    Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 7:730–737CrossRefGoogle Scholar
  3. 3.
    Kollet O, Peled A, Byk T, Ben Hur H, Greiner D, Shultz L, Lapidot T (2000) beta2 microglobulin-deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function. Blood 10:3102–3105Google Scholar
  4. 4.
    Vargaftig J, Taussig DC, Griessinger E, Anjos-Afonso F, Lister TA, Cavenagh J, Oakervee H, Gribben J, Bonnet D (2012) Frequency of leukemic initiating cells does not depend on the xenotransplantation model used. Leukemia 26:858–860PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Valent P, Bonnet D, De MR, Lapidot T, Copland M, Melo JV, Chomienne C, Ishikawa F, Schuringa JJ, Stassi G, Huntly B, Herrmann H, Soulier J, Roesch A, Schuurhuis GJ, Wohrer S, Arock M, Zuber J, Cerny-Reiterer S, Johnsen HE, Andreeff M, Eaves C (2012) Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer 11:767–775CrossRefGoogle Scholar
  6. 6.
    Hope KJ, Jin L, Dick JE (2004) Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 7:738–743CrossRefGoogle Scholar
  7. 7.
    Pearce DJ, Taussig D, Zibara K, Smith LL, Ridler CM, Preudhomme C, Young BD, Rohatiner AZ, Lister TA, Bonnet D (2006) AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML. Blood 3:1166–1173Google Scholar
  8. 8.
    Groen RW, Noort WA, Raymakers RA, Prins HJ, Aalders L, Hofhuis FM, Moerer P, van Velzen JF, Bloem AC, van Kessel B, Rozemuller H, van Binsbergen E, Buijs A, Yuan H, de Bruijn JD, de Weers M, Parren PW, Schuringa JJ, Lokhorst HM, Mutis T, Martens AC (2012) Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood 3:e9–e16CrossRefGoogle Scholar
  9. 9.
    Rizo A, Vellenga E, de Haan G, Schuringa JJ (2006) Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum Mol Genet 15(2):R210–R219PubMedCrossRefGoogle Scholar
  10. 10.
    Gartner S, Kaplan HS (1980) Long-term culture of human bone marrow cells. Proc Natl Acad Sci U S A 8:4756–4759CrossRefGoogle Scholar
  11. 11.
    Eaves CJ, Casman JD, Eaves AC (1991) Methodology of long-term culture of human hemopoietic cells. J Tiss Cult Meth 13:55–62CrossRefGoogle Scholar
  12. 12.
    Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM (1989) Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 5:1563–1570Google Scholar
  13. 13.
    Coulombel L, Eaves AC, Eaves CJ (1983) Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 2:291–297Google Scholar
  14. 14.
    Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ (1990) Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci U S A 9:3584–3588CrossRefGoogle Scholar
  15. 15.
    Gartner S, Kaplan HS (1981) Long-term culture of normal and leukemic human bone marrow. Haematol Blood Transfus 26:276–288PubMedGoogle Scholar
  16. 16.
    Scholzel C, Lowenberg B (1985) Stimulation of proliferation and differentiation of acute myeloid leukemia cells on a bone marrow stroma in culture. Exp Hematol 7:664–669Google Scholar
  17. 17.
    Ailles LE, Gerhard B, Hogge DE (1997) Detection and characterization of primitive malignant and normal progenitors in patients with acute myelogenous leukemia using long-term coculture with supportive feeder layers and cytokines. Blood 7:2555–2564Google Scholar
  18. 18.
    Sutherland HJ, Blair A, Zapf RW (1996) Characterization of a hierarchy in human acute myeloid leukemia progenitor cells. Blood 11:4754–4761Google Scholar
  19. 19.
    van Gosliga D, Schepers H, Rizo A, van der Kolk D, Vellenga E, Schuringa JJ (2007) Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells. Exp Hematol 10:1538–1549CrossRefGoogle Scholar
  20. 20.
    Schuringa JJ, Schepers H (2009) Ex vivo assays to study self-renewal and long-term expansion of genetically modified primary human acute myeloid leukemia stem cells. Methods Mol Biol 287–300Google Scholar
  21. 21.
    Han L, Wierenga AT, Rozenveld-Geugien M, van de Lande K, Vellenga E, Schuringa JJ (2009) Single-cell STAT5 signal transduction profiling in normal and leukemic stem and progenitor cell populations reveals highly distinct cytokine responses. PLoS One 11:e7989CrossRefGoogle Scholar
  22. 22.
    Rizo A, Horton SJ, Olthof S, Dontje B, Ausema A, van Os R, van den Boom V, Vellenga E, de Haan G, Schuringa JJ (2010) BMI1 collaborates with BCR-ABL in leukemic transformation of human CD34+ cells. Blood 22:4621–4630CrossRefGoogle Scholar
  23. 23.
    Rizo A, Dontje B, Vellenga E, de Haan G, Schuringa JJ (2008) Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1. Blood 5:2621–2630CrossRefGoogle Scholar
  24. 24.
    Rizo A, Olthof S, Han L, Vellenga E, de Haan G, Schuringa JJ (2009) Repression of BMI1 in normal and leukemic human CD34(+) cells impairs self-renewal and induces apoptosis. Blood 8:1498–1505CrossRefGoogle Scholar
  25. 25.
    Bonardi F, Fusetti F, Deelen P, van Gosliga D, Vellenga E, Schuringa JJ (2013) A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers. Mol Cell Proteomics 3:626–637CrossRefGoogle Scholar
  26. 26.
    Rozenveld-Geugien M, Baas IO, van Gosliga D, Vellenga E, Schuringa JJ (2007) Expansion of normal and leukemic human hematopoietic stem/progenitor cells requires rac-mediated interaction with stromal cells. Exp Hematol 5:782–792CrossRefGoogle Scholar
  27. 27.
    Schepers H, Wierenga AT, van Gosliga D, Eggen BJ, Vellenga E, Schuringa JJ (2007) Reintroduction of C/EBPalpha in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis. Blood 4:1317–1325CrossRefGoogle Scholar
  28. 28.
    Schepers H, van Gosliga D, Wierenga AT, Eggen BJ, Schuringa JJ, Vellenga E (2007) STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood 8:2880–2888CrossRefGoogle Scholar
  29. 29.
    Woolthuis CM, Han L, Verkaik-Schakel RN, van Gosliga D, Kluin PM, Vellenga E, Schuringa JJ, Huls G (2012) Downregulation of MEIS1 impairs long-term expansion of CD34(+) NPM1-mutated acute myeloid leukemia cells. Leukemia 26:848–853PubMedCrossRefGoogle Scholar
  30. 30.
    Barabe F, Kennedy JA, Hope KJ, Dick JE (2007) Modeling the initiation and progression of human acute leukemia in mice. Science 5824:600–604CrossRefGoogle Scholar
  31. 31.
    Wei J, Wunderlich M, Fox C, Alvarez S, Cigudosa JC, Wilhelm JS, Zheng Y, Cancelas JA, Gu Y, Jansen M, Dimartino JF, Mulloy JC (2008) Microenvironment determines lineage fate in a human model of MLL-AF9 leukemia. Cancer Cell 6:483–495CrossRefGoogle Scholar
  32. 32.
    Horton SJ, Jaques J, Woolthuis C, van Dijk J, Mesuraca M, Huls G, Morrone G, Vellenga E, Schuringa JJ (2013) MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny. Leukemia 27:1116–1126PubMedCrossRefGoogle Scholar
  33. 33.
    Chalandon Y, Jiang X, Christ O, Loutet S, Thanopoulou E, Eaves A, Eaves C (2005) BCR-ABL-transduced human cord blood cells produce abnormal populations in immunodeficient mice. Leukemia 3:442–448CrossRefGoogle Scholar
  34. 34.
    Chung KY, Morrone G, Schuringa JJ, Wong B, Dorn DC, Moore MA (2005) Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis. Blood 1:77–84CrossRefGoogle Scholar
  35. 35.
    Fatrai S, van Gosliga D, Han L, Daenen SM, Vellenga E, Schuringa JJ (2011) KRAS(G12V) enhances proliferation and initiates myelomonocytic differentiation in human stem/progenitor cells via intrinsic and extrinsic pathways. J Biol Chem 8:6061–6070CrossRefGoogle Scholar
  36. 36.
    Schuringa JJ, Wu K, Morrone G, Moore MA (2004) Enforced activation of STAT5A facilitates the generation of embryonic stem-derived hematopoietic stem cells that contribute to hematopoiesis in vivo. Stem Cells 7:1191–1204CrossRefGoogle Scholar
  37. 37.
    Chung KY, Morrone G, Schuringa JJ, Plasilova M, Shieh JH, Zhang Y, Zhou P, Moore MA (2006) Enforced expression of NUP98-HOXA9 in human CD34(+) cells enhances stem cell proliferation. Cancer Res 24:11781–11791CrossRefGoogle Scholar
  38. 38.
    Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, Metzeler KH, Poeppl A, Ling V, Beyene J, Canty AJ, Danska JS, Bohlander SK, Buske C, Minden MD, Golub TR, Jurisica I, Ebert BL, Dick JE (2011) Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 9:1086–1093CrossRefGoogle Scholar
  39. 39.
    Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Luke T, Lillington D, Oakervee H, Cavenagh J, Agrawal SG, Lister TA, Gribben JG, Bonnet D (2010) Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(−) fraction. Blood 10:1976–1984CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Pallavi Sontakke
    • 1
  • Marco Carretta
    • 1
  • Marta Capala
    • 1
  • Hein Schepers
    • 1
  • Jan Jacob Schuringa
    • 1
    Email author
  1. 1.Department of Experimental HematologyUniversity Medical Center Groningen, University of GroningenGroningenThe Netherlands

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