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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

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Hematopoietic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((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.

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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).

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Authors and Affiliations

  1. Department of Experimental Hematology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands

    Pallavi Sontakke, Marco Carretta, Marta Capala, Hein Schepers & Jan Jacob Schuringa

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  1. Pallavi Sontakke
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  2. Marco Carretta
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  3. Marta Capala
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  4. Hein Schepers
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  5. Jan Jacob Schuringa
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Corresponding author

Correspondence to Jan Jacob Schuringa .

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Editors and Affiliations

  1. Department of Pediatrics, Aflac Cancer and Blood Disorders Center Emory University School of Medicine, Atlanta, Georgia, USA

    Kevin D. Bunting

  2. Department of Pediatrics, Emory University School of Medicine Aflac Cancer & Blood Disorders Ctr, Atlanta, Georgia, USA

    Cheng-Kui Qu

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Sontakke, P., Carretta, M., Capala, M., Schepers, H., Schuringa, J.J. (2014). Ex Vivo Assays to Study Self-Renewal, Long-Term Expansion, and Leukemic Transformation of Genetically Modified Human Hematopoietic and Patient-Derived Leukemic Stem Cells. In: Bunting, K., Qu, CK. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Biology, vol 1185. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1133-2_13

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  • DOI: https://doi.org/10.1007/978-1-4939-1133-2_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1132-5

  • Online ISBN: 978-1-4939-1133-2

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