We have recently demonstrated the presence of hematopoietic stem cells(HSC) in the murine yolk sac (YS) at day 9 postcoitus(pc) (PNAS 94:6776-6780). We now wished to isolate and characterize HSC from the YS and to explore the possible existence of a consistent phenotype of HSC throughout ontogeny. We utilized commercially available monoclonal antibodies to c-Kit, CD38, TER-119, MAC-1, GR-1, B220, CD4, and CD8 to isolate c-Kit+ lineage- CD38 +/- adult murine bone marrow (BM) and c-Kit+ TER 119- CD38 +/- day 9 pc YS cells. Cells were stained with propidium iodide and analyzed for position in the cell cycle based upon DNA content. Cells were cultured in an HPP-CFC assay and colonies counted on day 14 or were transplanted into congenic recipient mice. Lethally irradiated adult congenic C57BL/6J and Busulfan conditioned newborn mice were injected with 200 donor and 2 × 105 competitor bone marrow derived mononuclear cells. Percent donor chimerism was determined by Gpi-1 isoenzyme analysis of peripheral blood at several time points after transplant. Multilineage leukocyte analysis of peripheral blood at 6 months post-transplant revealed identical chimerism compared to red cell analysis. HPP-CFC were enriched in cKit+Lin-CD38+ YS (362 ± 45) and BM (373± 75) sorted cell populations compared to cKit+lin- CD38- YS (25± 11) and BM (53 ± 11) cells. More YS cKit+Lin-CD38+ cells were found in cycle (45% in S/G2+M) compared to BM CD38+ cells (30% in S/G2+M). All long-term repopulating ability was present in donor YS and BM cKit+Lin-CD38+ cells (40% and 41% mean donor chimerism respectively at 6 months compared to 0% and 0% for cKit+Lin-CD38- BM and YS cells). We have shown that repopulating HSC expressing CD38 can be isolated from embryonic and adult hematopoietic sites. While HSC from both sites repopulate donor marrow, some differences in biology exist in cells isolated at these stages of ontogeny.
