Constitutive Expression of the ABCG2 (BCRP), the Molecular Determinant of the Side Population, Increases the Proliferative Potential of Human Clonogenic Progenitors and Supports Human Myeloid Engraftment in the NOD/SCID Mouse Model.

Adult hematopoietic stem cells can be identified by the ability to rapidly efflux the Hoechst 33342 dye and consequently produce a characteristic side population (SP) phenotype. ABCG2 (Human Breast Cancer Resistance Protein, BCRP) is the molecular determinant of the SP phenotype. We have demonstrated previously that the SP phenotype together with the expression of CD34 and lack of CD38 distinguishes between normal and leukemic stem cells in patients with acute myeloid leukemia (AML), suggesting a role of this protein in early human hematopoiesis. To test this, normal highly purified human CD34⁺ cord blood cells were transduced retrovirally by ABCG2/YFP and analyzed for their in vitro and in vivo behaviour. In vitro constitutive expression of ABCG2 doubled the number of the most immature CFU-GEMM type colonies in the CFC assays (n=12; p< 0.002). Furthermore, the protein enhanced the replating capacity of primary colonies with a mean 3.0 fold increase in the number of 2^nd colonies (n=9; p< 0.01), indicating a substantial enhancement of the proliferative potential of clonogenic progenitors by constitutive ABCG2 expression. In contrast, ABCG2 did not induce any major increase in the frequency of LTC-IC compared to the YFP control after 5 days as assessed by limiting dilution LTC-IC (1 LTC-IC per 3911 cells and 1 LTC-IC per 3641 cells, respectively). To study the impact of ABCG2 on human progenitor cells in vivo NOD/SCID mice were injected with highly purified ABCG2/YFP⁺ cells and analyzed 8 weeks after transplantation for human engraftment. Although mice in the ABCG2 group received less transduced cells than the control (on average 1.2 x 10⁵ versus 3.7 x 10⁵ per mouse, respectively), they showed significant higher engraftment compared to the control group (6.1 x 10⁷ transduced cells (4.3–8.2) versus 4.2 x 10⁷ (3.2–5.7) per mouse, respectively; p<0.04). Mice that received ABCG2-transduced cells showed a 4.6fold increase in the number of engrafted CD34⁺ progenitor cells (1.4x 10⁷ CD34⁺CD45⁺ vs 6.5x 10⁶; p<0.05). In addition, ABCG2 expression resulted in 2.2-fold increase of c-KIT⁺ cells (6.1x10⁶ cells vs. 2.8 x 10⁶ cells in the control arm; p< 0.02) indicating that the constitutive expression of ABCG2 enhanced the number of human primitive progenitor cells. ABCG2 expression was also associated with an expansion in the CD15⁺ /CD33⁺ human myeloid compartment: in the control mice 1.1 x 10⁷ human transduced myeloid cells (CD15⁺) were detected per mouse compared to 2.6 x 10⁷ in the ABCG2 group 8 weeks post transplant (p<0.05) whereas the human CD19⁺ lymphoid compartment was not changed. This resulted in an inversion of the ratio of engrafted CD19⁺/CD15⁺ human lymphoid/myeloid cells (mean of 0.5 for ABCG2 vs 1.1 in the control; p<0.03). Furthermore, constitutive expression of ABCG2 promoted erythroid differentiation with a 3.6fold increase in glycophorin A expressing erythroid cells (9 x 10⁶ vs 2.5 x 10⁶ GlyA⁺ cells in the control; p < 0.003). Taken together, our data characterize ABCG2 as a previously unrecognized potent positive regulator of primitive hematopoietic cell growth in vitro and in vivo and extend our so far limited knowledge about human stem cell regulation by this ABC transporter.