Discerning the role of PU.1 and GFP in adult hematopoietic stem cells

Understanding the role of PU.1 in adult hematopoiesis has previously not been possible due to the embryonic lethality of the PU.1 −/− mouse. In order to overcome the embryonic lethal mutation, we have targeted the PU.1 locus with an inducible form of PU.1, PU:ERT, which is regulated at the protein level in response to tamoxifen (OHT). We sought to reconstitute adult hematopoiesis with PU.1 in the presence of OHT and then withdrawal PU.1 in adulthood to determine the effect of PU.1 withdrawal on the adult hematopoietic stem cell and mature lineages within the blood. In order to establish adult hematopoiesis, we transplanted PU:ERT +/+ fetal livers into adult, irradiated recipients congenic for Ly5 that were previously implanted with tamoxifen time-release pellets. We found that PU.1 is necessary for the establishment of adult hematopoiesis within the bone marrow. Due to the nature of the regulation of PU.1 by the OHT in vivo, we were unable to determine the role of PU.1 in sustaining mature lymphopoiesis and myelopoiesis. In addition, using a similar hematopoietic stem cell transplant model, we sought to determine if stem cell plasticity, namely generation of new endothelium, occurred with or without fusion and if the GFP level of the HSC played a role in this process. We found that GFP levels in the HSC do not seem to alter the ability of the HSC to engraft an irradiated host, nor do they correspond to cell cycle of the HSC, however, the role of GFP level in stem cell plasticity was not determined. Further study of adult HSCs and adult hematopoiesis is necessary to elucidate the potential of the HSC and HSC progenitors as possible sources of cell-based therapy