Model systems for examining effects of leukemia-associated oncogenes in primary human CD34+ cells via retroviral transduction

Abstract

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.

Fig. 1

Overview of the model system for retroviral transduction of human CD34+ cells. (a) Human CD34+ cells are purified by magnetic selection, and purity is confirmed by flow cytometric analysis. (b) A transduction of human CD34+ cells is shown, with a non-transduced control. GFP expression is visualized by flow cytometry.

Fig. 2

Overview of the in vitro analysis of retrovirally transduced human CD34+ cells. (A) Representative long-term myeloid cultures (6 weeks) of control CB cells and cells expressing the AML1-ETO oncogene (AE). This phenotype is preserved throughout the 6–8 months of growth. (B) Example of a cobblestone area that forms upon coculture of human CD34+ cells with the MS-5 stroma cell line. (C) B cells can be expanded from the transduced CD34+ cultures upon coculture with MS-5 and the cytokines Flt3L, SCF and IL-7. Shown is a CBFB-SMMHC-transduced culture that was 12 weeks posttransduction. Cells were cultured on MS-5 for an additional 4 weeks. A fraction of the CD19+ cells also express the CD20 B-cell marker. (D) A clonogenic methylcellulose assay is shown, with representative BFU-E (red cell colony) and CFU-GM (myeloid colony) that result from the growth of a single cell. (E, F) Expression of a fusion oncogene frequently leads to loss of clonogenic potential immediately upon transduction, and this effect is due to a G0/G1 cell cycle block for cells expressing the inv16 fusion protein CBFB-SMMHC.

Fig. 3

Overview of the in vivo analysis of retrovirally transduced human CD34+ cells. (A, B) Analysis of the bone marrow of a NOD/SCID-β2M^−/− mouse that was injected with control transduced (MIGR1) and CBFB-SMMHC-transduced (inv16) cells. 8.5 weeks after injection, the human graft in the bone marrow of the mouse was readily detectable, and GFP+ cells were present in both mice. Most of the human cells in the control mouse were CD19+ B-cells, as is typical, but for the CBFB-SMMHC-expressing cells, there is a loss of the B-cell fraction, and essentially all of the cells express the myeloid marker CD13.