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Purification and characterization of murine long-term lympho-myeloid repopulating hemopoietic stem cells Szilvassy, Stephen Joseph


The hemopoietic system Is organized as a hierarchy of hemopoietic cell populations distinguished by differences in their proliferation and differentiation potential. Studies using short-term in vitro and in vivo assays based on colony formation in semi-solid medium, or in the spleens of lethally irradiated mice, respectively, have shown that these procedures detect primarily lineage-restricted progenitor types and have provided much information about the characteristics and regulation of such cells. Assessment of lymphoid and myeloid tissue reconstitution after more prolonged periods following transplantation has established the existence of a more primitive stem cell type; however, the retrospective nature of these complex analyses has Impeded characterization and purification of these cells. My first objective was to develop a procedure for the selective isolation of stem cells with short-term in vitro and in vivo multilineage differentiation potential. For this I devised a single-step, four-parameter fluorescence activated cell sorting procedure In which cells were selected according to their forward and orthogonal light-scattering properties, and their surface expression of the Thy-1 and H-2K antigens. Application of this procedure to marrow cells from mice treated 4 days previously with 150 mg/kg of 5-fluorouracil showed that it could be used to sort a subpopulation that was enriched 100-fold in CFU-GEMM and in which 1 in 4 cells was a day 12 CFU-S. To determine the extent to which stem cells with long-term lympho-myeloid repopulating potential had been copurified, I undertook to develop a quantitative procedure that might allow this primitive cell population to be measured and hence characterized on a routine basis. This required an assay that would detect donor-derived hemopoiesis exclusively, and that was sensitive enough for the detection of limiting numbers of cells with long-term lympho-myeloid repopulating potential. This was shown to be possible using a competitive repopulation assay in which lethally irradiated female recipients were transplanted with male "test" cells together with a second suspension of female cells with adequate short-term repopulating activity but greatly diminished long-term repopulating potential. These sex differences were then used to specifically identify the 5 week progeny of stem cells in the test suspension. Assessment of the sorted day 4 5-FU marrow population revealed that it was capable of repopulating all hemopoietic organs after transplantation and that an enrichment of 30-fold over unseparated, 5-FU-treated marrow had been achieved. My second objective was to determine whether the competitive long-term lymphoid and myeloid repopulation obtained with these sorted cells was due to the activity of Individual stem cells with a dual potential for lymphopoiesis and myelopoiesis. For this I used retroviral-infection to uniquely mark sorted cells In vitro, and then transplanted them in sufficiently low numbers to allow individual regenerated clones to be detected and analyzed. In some mice, distribution of cells with the same unique integration marker in different lymphoid and myeloid cell populations established the presence of lympho-myeloid stem cells in the original sorted population. In addition, clones with restricted tissue distributions were also documented. My final objective was to investigate whether the competitive repopulation assay was in fact able to serve as a procedure for the exclusive quantitation of long-term lympho-myeloid repopulating stem cells. A limiting dilution approach was used to compare the frequency of hemopoietic stem cells (competitive repopulating units, CRU) in marrow obtained from a variety of sources, using >20% repopulation by male cells at 5 or 10 weeks post-transplantation as the end point. The results obtained were largely independent of the time of analysis, and whether repopulation of recipient marrow or thymus was evaluated, suggesting that either can be used in this assay to quantitate a hemopoietic stem cell with the potential to regenerate both lymphoid and myeloid systems. These studies have provided procedures for the detection, quantitation and selective enrichment of the most primitive stem cells in the murine hemopoietic system which have competitive long-term lympho-myeloid repopulating ability. The availability of these procedures should facilitate the development of additional purification steps leading to the isolation of these cells as homogeneous suspensions, and their further use as targets for retrovirus-medilated gene transfer to determine the genetic basis of their activation, determination and neoplastic transformation.

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