Abstract
The GATA-2 transcription factor is required for the development and regulation of blood stem cells as judged by the embryonic lethal, “bloodless” phenotype of mice lacking GATA-2 and the high levels of GATA-2 present in adult stem cells which necessarily decline with differentiation into the various blood lineages. Our previous studies showing that sustained, high level GATA-2 expression in adult murine stem cells blocks both stem cell amplification and differentiation in vivo (Persons, Blood 93:488, 1999) suggest that the fate of primitive repopulating cells is regulated by the level of GATA-2. To further investigate the stem cell regulatory activity of GATA-2, we developed a retroviral vector encoding a tamoxifen inducible, GATA-2/estrogen receptor chimeric protein (G2-ER). In the presence of tamoxifen, murine hematopoietic progenitors transduced with the G2-ER/GFP retroviral vector generated only 6% ± 5% (n=5) of the number of myeloid colonies formed in the absence of tamoxifen, consistent with our previous findings and confirming the functional activity of the fusion protein. Surprisingly, in the absence of tamoxifen, G2-ER colonies grew significantly larger in size than control colonies. In addition, G2-ER transduced bone marrow (BM) cells out-competed mock-transduced cells in liquid culture. Control GFP or G2-ER transduced BM cells were diluted with mock-transduced BM cells at a 1:10 ratio. At 7 days, the control culture was 10% GFP+ and the G2-ER culture was 17% positive. However, by day 11 the control culture remained 10% GFP+, while the G2-ER culture increased to 87% GFP+. Thus far, G2-ER transduced cells can be expanded for up to 12 weeks in culture and, in contrast to control cultures which are morphologically comprised of mast cells after the same time in culture, the G2-ER cells are predominantly immature appearing myeloid cells and blasts. FACS analysis confirmed the myeloid phenotype of the expanded G2-ER cells (83% CD11b+, c-kit−), while the control cells showed a mast cell phenotype (100% c-kit+, IgE receptor+). The G2-ER pro-proliferative effect was also observed in transplanted mice since there was a significant repopulation advantage of G2-ER myelo-erythroid cells over untransduced cells. Mice transplanted with G2-ER transduced BM mixed 1:1 with untransduced marrow at 12 weeks post-transplant showed 82% ± 6 GFP+ Gr-1+ cells (n=10) and 57% ± 4 RBCs compared to 28% ± 17 (n=7) and 10% ± 5 in GFP: Mock transplanted control mice. These differences were not due to differences in the transduction rate of primitive repopulating cells as judged by gene transfer into the Sca+ Lin− cells contained in the grafts. Interestingly, there was a lack of B lymphoid development from transduced stem cells in G2-ER transplanted mice at 12 weeks since few peripheral blood B220+ cells co-expressed GFP (8% ± 2), compared to controls (58% ± 14). Similarly, development of transduced T cells was also delayed but showed a rebound with low expressing GFP+ cells at late time points. Analysis of bone marrow, spleen, and thymus of transplanted mice confirmed the poor development of lymphoid cells and showed that there was not a developmental arrest in these organs. Since it is likely that small amounts of G2-ER are active in the absence of tamoxifen, these data suggest that the concentration of GATA-2 in specific lineage precursors is critical in regulating the expansion and differentiation into the different blood lineages. Studies to identify GATA-2 interactions with other transcription factors may uncover the mechanisms that regulate the differentiation of specific lineage precursors.
GSK3β, a Master Kinase in the Regulation of Adult Stem Cell Behavior