Levels of the c-Myb Protein Indicate Repopulating Capacity in Long-Term Hematopoietic Stem Cells

Abstract 1196

c-Myb is a transcription factor essential for the proliferation of hematopoietic cells: conventional c-myb deficient mice died around E14 when their hematopoietic progenitors/stem cells fail to proliferate in the fetal livers. Recently, c-myb has also been reported to be crucial for the differentiation of hematopoietic progenitors. We have previously reported that the differentiation into erythrocytes, megakaryocytes and B-lymphocytes is regulated by c-myb levels utilizing ES cell in vitro differentiation combined with a tetracycline-inducible gene expression system. The gene-altered c-myb mice, such as knockdown or conditional knockout mice in the hematopoietic cell lineages, showed that c-myb controlled hematopoietic stem cells (HSCs). In order to examine the levels of the c-Myb protein in HSCs, we established c-Myb reporter mice in which the EGFP cDNA was linked to the coding sequence of the c-myb gene (c-MybEGFP). Homozygous c-MybEGFP mice, showing normal hematopoiesis, expressed EGFP in hematopoietic progenitors. EGFP⁺ cells were observed in most long-term (LT) HSCs (90–95%), which were defined as CD34⁻ Lin⁻ Sca-1⁺c-Kit^high cells (34LSKs), CD150⁺CD48⁻LSKs and side-population LSKs. To evaluate c-Myb function in LT-HSCs, we transplanted 100 cells of EGFP^low and EGFP^high of 34LSKs into irradiated mice along with competitor cells (0×10⁶ cells). Both LT-HSC populations presented multilineage repopulating capacity over 20 weeks. In addition, the EGFP^low cells indicated higher chimerism in the total peripheral blood than the EGFP^high cells at any given time point. The contribution of the EGFP^low-derived cells in the peripheral blood of the recipient mice increased over time whereas EGFP^high progeny gradually decreased over time. Under a stringent transplantation condition (30 donor cells with 0.4×10⁶ competitor cells), 83.3% of the recipients that received the EGFP^low34LSK showed donor-derived progeny while the EGFP^high were lower (20.0%) 8 weeks after transplantation. At Week 12, all the recipients with the EGFP^low34LSKs demonstrated donor-derived progeny; however, EGFP^high 34LSKs-derived cells disappeared totally in all the transplants. These results suggest that the EGFP^low and the EGFP^high cells in LT-HSCs possess distinct repopulating capacity: the EGFP^low cells are high and the EGFP^high cells are low. To investigate the relationship between the EGFP^low and the EGFP^high LT-HSC, we examined EGFP expression levels in the recipient mice grafted EGFP^low34KSL at least 24 weeks after transplantation. EGFP^low34LSK generated EGFP^high cells in the donor-derived 34LSK population in the recipient mice, suggesting the possibility that the EGFP^low LT-HSCs support the production of the EGFP^high LT-HSCs. In conclusion, we found that the expression levels of c-Myb protein subdivide LT-HSC population in correspondence with their respective multilineage repopulating capacities.