THE MPL-LIGAND IS INVOLVED IN THE GROWTH-PROMOTING ACTIVITY OF THE MURINE STROMAL CELL-LINE MS-5 ON ES CELL-DERIVED HEMATOPOIESIS

We investigated the ability of the murine stromal cell line MS-5 to en hance the hematopoietic potential of embryonic stem (ES) cells. The pr esence of increasing concentrations of MS-5 cells during the different iation of ES cells into embryoid bodies (EBs) resulted in a positive d ose effect on the efficiency of EB development. Moreover, the number o f myeloid progenitors derived from EBs at days 6 and 10 of differentia tion significantly increased. This increase resulted from an elevation of both the proportions of positive EBs (EBs containing at least one progenitor each) and the progenitor cell content per positive EB. The stimulatory activity of MS-5 cells affected all types of myeloid proge nitors except erythroid progenitors, which were depressed. However, th e relative numbers of ES-derived granulocyte-macrophage progenitors (c olony-forming units granulocyte/macrophage [CFU-GM], - macrophage [CFU -M], and -granulocyte [CFU-GI]) and of mixed cell colonies were unchan ged. In contrast, the incidence of megakaryocytic progenitors (colony- forming units-megakaryocyte [CFU-MK]) was significantly increased, tha t of erythroid progenitors (burst-forming units-erythroid [BFU-E]) was concomitantly decreased, and the total numbers of both progenitor typ es remained constant. Addition of Mpl-ligand (Mpl-L; thrombopoietin) d uring the growth of EBs was found to mimic the effect of the MS-5 cell line on the output of progenitor cells. No effect of Mpl-L on the eff iciency of EB formation was observed. In addition, supplementation of cultures with sufficient soluble Mpl to abrogate Mpl-L activity result ed in the reversion of the quantitative and qualita tive effects of MS -5 cells on progenitor cell formation but not on the efficiency of EB formation. Together, these data indicate two major effects and two lev els of action of the MS-5 cell line on hematopoietic differentiation o f ES cells. First, the cell line acts before hematopoietic determinati on, promoting the plating efficiency of ES cells via mechanisms that r emain to be clarified. Second, at a later stage of differentiation, th e MS-5 cells promote hematopoiesis within EBs. Mpl-L appears to be one of the components that confer this latter ability on the MS-5 cell li ne.