Model Systems for Examining Effects of Leukemia Associated Oncogenes in Primary Human CD34+ Cells via Retroviral Transduction

Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 538:263-85. DOI: 10.1007/978-1-59745-418-6_13
Source: PubMed


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.

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Available from: Mark Wunderlich
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    • "To evaluate the involvement of WT RUNX1 in the development of A-E-mediated preleukemic cell phenotype, we used a preleukemic cell model (Mulloy et al., 2002; Wunderlich and Mulloy, 2009) of human CD34 + progenitor cells transduced by A-E expressing lentivirus (Millington et al., 2009). Transfection of CD34 + /A-E cells with siRNA against RUNX1 (Figure S6) resulted in reduced expression of RUNX1 associated with an increased proportion of Annexin-V + positive cells as compared with cells transfected with control NT siRNA (Figure 7C and 7D). "
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    • "Cells were subsequently plated into (1) H4100 methylcellulose (Stem Cell Technologies) containing 20% heat-inactivated FCS, 10 À4 M b-ME, 20% IMDM, 2 mM L-glutamine, and 10 ng/ml each of human recombinant SCF, IL-6, IL-3, G-CSF (PeproTech EC), and 6 U/ml erythropoietin (EPO) (R&D Systems, Abingdon, UK) (Wunderlich and Mulloy, 2009); and (2) coculture with irradiated MS5 stromal cells in long-term culture (LTC) medium (alpha-MEM supplemented with heat-inactivated 12.5% FCS, heat-inactivated 12.5% horse serum [Sigma-Aldrich], 2 mM L-glutamine, 57.2 mM b-ME, and 20 ng/ml each of human recombinant TPO, IL-3, and G-CSF) (Schuringa and Schepers, 2009). Cultures were kept at 37 C and 5% CO 2 and replated on new MS5 stroma every week. "
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