EVIDENCE OF BOTH ONTOGENY AND TRANSPLANT DOSE-REGULATED EXPANSION OF HEMATOPOIETIC STEM-CELLS IN-VIVO

Recent assessment of the long-term repopulating activity of defined su bsets of hematopoietic cells has offered new insights into the charact eristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their s elf-renewal in vivo. We have now exploited the ability to quantitate t hese cells using the competitive repopulating unit (CRU) assay to iden tify the role of both intrinsic (ontological) and extrinsic (transplan ted dose-related) variables that may contribute to the regulation of C RU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liv er (FL) or the bone marrow (BM) of adult mice injected 4 days previous ly with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-sple en numbers irrespective of the number or origin of cells initially tra nsplanted, In contrast, regeneration of Ly5.1(+) donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult B M. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in reci pients of FL and adult BM transplantation, respectively, with an accom panying maximum CRU amplification of 150-fold for recipients of FL cel ls and 15-fold for recipients of adult BM cells. Interestingly, the ex tent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulato ry mechanisms to prematurely limit stem cell self-renewal ability. Pro viral integration analysis of mice receiving retrovirally transduced B M cells confirmed regeneration of totipotent lymphomyeloid repopulatin g cells and provided evidence for a greater than 300-fold clonal ampli fication of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sour ces that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell am plification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage f or attempts to enhance CRU regeneration by administration of agents th at may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic re generative capacity. (C) 1996 by The American Society of Hematology.