Summary
The use of primary human cells to model cancer initiation and progression is now within the grasp of investigators. It has been nearly a decade since the first defined genetic elements were introduced into primary human epithelial and fibroblast cells to model oncogenesis. This approach has now been extended to the hematopoietic system, with the first described experimental transformation of primary human hematopoietic cells. Human cell model systems will lead to a better understanding of the species and cell type specific signals necessary for oncogenic initiation and progression, and will allow investigators to interrogate the cancer stem cell hypothesis using a well-defined hierarchical system that has been studied for decades. The molecular and biochemical link between self-renewal and differentiation can now be experimentally approached using primary human cells. In addition, the models that result from these experiments are likely to generate highly relevant systems for use in identification and validation of potential therapeutic targets as well as testing of small molecule therapeutics. We describe here the methodologies and reagents that are used to examine the effects of leukemia fusion protein expression on primary human hematopoietic cells, both in vitro and in vivo.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sharpless NE, Depinho RA. (2006). The mighty mouse: Genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov;5(9):741–54.
Rangarajan A, Weinberg RA. (2003). Opinion: Comparative biology of mouse versus human cells: Modelling human cancer in mice. Nat Rev Cancer;3(12):952–9.
Rangarajan A, Hong SJ, Gifford A, Weinberg RA. (2004). Species- and cell type-specific requirements for cellular transformation. Cancer Cell;6(2):171–83.
Drayton S, Peters G. (2002). Immortalisation and transformation revisited. Curr Opin Genet Dev;12(1):98–104.
Smogorzewska A, de Lange T. (2002). Different telomere damage signaling pathways in human and mouse cells. Embo J ;21(16):4338–48.
Hamad NM, Elconin JH, Karnoub AE, et al. (2002). Distinct requirements for Ras oncogenesis in human versus mouse cells. Genes Dev;16(16):2045–57.
Lim KH, Baines AT, Fiordalisi JJ, et al. (2005). Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell;7(6):533–45.
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. (1999). Creation of human tumour cells with defined genetic elements. Nature;400(6743):464–8.
Hahn WC, Weinberg RA. (2002). Rules for making human tumor cells. N Engl J Med;347(20):1593–603.
Pereira DS, Dorrell C, Ito CY, et al. (1998). Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells. Proc Natl Acad Sci U S A;95(14):8239–44.
Grignani F, Valtieri M, Gabbianelli M, et al. (2000). PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype. Blood;96(4):1531–7.
Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Moore MA, Nimer SD. (2002). The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells. Blood;99(1):15–23.
Buske C, Feuring-Buske M, Antonchuk J, et al. (2001). Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo. Blood;97(8):2286–92.
Daga A, Podesta M, Capra MC, Piaggio G, Frassoni F, Corte G. (2000). The retroviral transduction of HOXC4 into human CD34(+) cells induces an in vitro expansion of clonogenic and early progenitors. Exp Hematol;28(5):569–74.
Barabe F, Kennedy JA, Hope KJ, Dick JE. (2007). Modeling the initiation and progression of human acute leukemia in mice. Science;316(5824):600–4.
Bowie MB, Kent DG, Dykstra B, et al. (2007). Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties. Proc Natl Acad Sci U S A;104(14):5878–82.
Holyoake TL, Nicolini FE, Eaves CJ. (1999). Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol;27(9):1418–27.
Kelly PF, Carrington J, Nathwani A, Vanin EF. (2001). RD114-pseudotyped oncoretroviral vectors. Biological and physical properties. Ann N Y Acad Sci;938:262–76; discussion 76–7.
Kelly PF, Vandergriff J, Nathwani A, Nienhuis AW, Vanin EF. (2000). Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. Blood;96(4):1206–14.
Hanawa H, Kelly PF, Nathwani AC, et al. (2002). Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther;5(3):242–51.
Wunderlich M, Krejci O, Wei J, Mulloy JC. (2006). Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability. Blood;108(5):1690–7.
Mulloy JC, Cammenga J, Berguido FJ, et al. (2003). Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element. Blood;102(13):4369–76.
Itoh K, Tezuka H, Sakoda H, et al. (1989). Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp Hematol;17(2):145–53.
Ito M, Hiramatsu H, Kobayashi K, et al. (2002). NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood;100(9):3175–82.
Nicolini FE, Cashman JD, Hogge DE, Humphries RK, Eaves CJ. (2004). NOD/SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration. Leukemia;18(2):341–7.
Feuring-Buske M, Gerhard B, Cashman J, Humphries RK, Eaves CJ, Hogge DE. (2003). Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors. Leukemia;17(4):760–3.
Haas DL, Case SS, Crooks GM, Kohn DB. (2000). Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. Mol Ther;2(1):71–80.
Sandrin V, Boson B, Salmon P, et al. (2002). Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates. Blood;100(3):823–32.
Wang J, Kimura T, Asada R, et al. (2003). SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection. Blood;101(8): 2924–31.
Yahata T, Ando K, Sato T, et al. (2003). A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow. Blood;101(8):2905–13.
Wei J, Wunderlich M, Fox C, Alvarez S, Cigudosa JC, Wilhelm JE, Zheng Y, Cancelas JA, Gu Y, Jansen M, DiMartino JF and Mulloy, JC (2008) Microenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia. Cancer Cell; 13 (6): 483–495.
Mulloy JC, Wunderlich M, Zheng Y, Wei J. (2008) Transforming Human Blood Stem and Progenitor Cells: A New Way Forward in Leukemia Modeling. Cell Cycle; 7(21): 57–52.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Wunderlich, M., Mulloy, J. (2009). Model Systems for Examining Effects of Leukemia Associated Oncogenes in Primary Human CD34+ Cells via Retroviral Transduction. In: Eric So, C.W. (eds) Leukemia. Methods in Molecular Biology™, vol 538. Humana Press. https://doi.org/10.1007/978-1-59745-418-6_13
Download citation
DOI: https://doi.org/10.1007/978-1-59745-418-6_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-989-5
Online ISBN: 978-1-59745-418-6
eBook Packages: Springer Protocols