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-Kithigh cells (34LSKs), CD150+CD48−LSKs and side-population LSKs. To evaluate c-Myb function in LT-HSCs, we transplanted 100 cells of EGFPlow and EGFPhigh 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 EGFPlow cells indicated higher chimerism in the total peripheral blood than the EGFPhigh cells at any given time point. The contribution of the EGFPlow-derived cells in the peripheral blood of the recipient mice increased over time whereas EGFPhigh 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 EGFPlow34LSK showed donor-derived progeny while the EGFPhigh were lower (20.0%) 8 weeks after transplantation. At Week 12, all the recipients with the EGFPlow34LSKs demonstrated donor-derived progeny; however, EGFPhigh 34LSKs-derived cells disappeared totally in all the transplants. These results suggest that the EGFPlow and the EGFPhigh cells in LT-HSCs possess distinct repopulating capacity: the EGFPlow cells are high and the EGFPhigh cells are low. To investigate the relationship between the EGFPlow and the EGFPhigh LT-HSC, we examined EGFP expression levels in the recipient mice grafted EGFPlow34KSL at least 24 weeks after transplantation. EGFPlow34LSK generated EGFPhigh cells in the donor-derived 34LSK population in the recipient mice, suggesting the possibility that the EGFPlow LT-HSCs support the production of the EGFPhigh 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.