CD34(-CELL LINES DERIVED FROM MURINE YOLK-SAC INDUCE THE PROLIFERATION AND DIFFERENTIATION OF YOLK-SAC CD34(+) HEMATOPOIETIC PROGENITORS() ENDOTHELIAL)

Embryonic hematopoiesis is initiated in part in the blood islands of t he yolk sac. Previous confocal microscopic analysis has shown that the CD34 antigen, a mucin-like cell surface glycoprotein that is expresse d by hematopoietic progenitors and all endothelial cells of the adult and embryo, is also found on a subset of luminal hematopoietic-like ce lls in the yolk sac blood islands as well as on the vascular endotheli um lining these early hematopoietic locations. We show here that, as i n all other hematopoietic sites thus far examined, immunoaffinity-puri fied CD34(+) nonadherent cells from murine yolk sacs contain the vast majority of erythroid and myeloid progenitor cell colony forming activ ity. To examine the developmental interactions between these CD34(+) h ematopoietic progenitor cells of the yolk sac and the CD34(+) yolk sec endothelium, we have immunaffinity-purified adherent endothelial cell s from day 10.5 yolk sacs using CD34 antiserum and produced cell lines by transformation with a retrovirus expressing the polyoma middle T a ntigen. Analysis of these cell lines for CD34, von Willebrand's factor , FLK 1 and FLT 1 expression, and capillary growth in Matrigel indicat es that they appear to be endothelial cells, consistent with their ori ginal phenotype in vivo. Coculture of yolk sac CD34(+) hematopoietic c ells on these endothelial cell lines results in up to a 60-fold increa se in total hematopoietic cell number after approximately 8 days. Anal ysis of these expanded hematopoietic cells showed that the majority we re of the monocyte/macrophage lineage. In addition, examination of the cultures showed the rapid formation of numerous cobblestone areas, a previously described morphologic entity thought to be representative o f early pluripotential stem cells. Scrutiny of the ability of these en dothelial cell lines to expand committed progenitor cells showed up to a sixfold increase in erythroid and myeloid colony-forming cells afte r 3 to 6 days in culture, consistent with the notion that these embryo nic endothelial cells mediate the expansion of these precursor cells. Polymerase chain reaction analyses showed that most of the cell lines produce FLK-2/FLT-3 ligand, stem cell factor, macrophage colony-stimul ating factor, leukemia-inhibitory factor, and interleukin 6 (IL-6), wh ereas there is a generally low or not measurable production of granulo cyte colony-stimulating factor, granulocyte-macrophage colony-stimulat ing factor, IL-1, IL-3, transforming growth factor beta-1, erythropoie tin, or thrombopoietin. The output of mature hematopoietic cells from these cocultures can be modified to include an erythroid population by the addition of exogenous erythropoietin. These data suggest that end othelial cell lines derived from the yolk sac provide an appropriate h ematopoietic environment for the expansion and differentiation of yolk sac progenitor cells into at least the myeloid and erythroid lineages . (C) 1995 by The American Society of Hematology.