IN-VITRO AND IN-VIVO DIFFERENTIATION INTO B-CELLS, T-CELLS, AND MYELOID CELLS OF PRIMITIVE YOLK-SAC HEMATOPOIETIC PRECURSOR CELLS EXPANDED GREATER-THAN-100-FOLD BY COCULTURE WITH A CLONAL YOLK-SAC ENDOTHELIAL-CELL LINE

The yolk sac, first site of hematopoiesis during mammalian development , contains not only hematopoietic stem cells but also the earliest pre cursors of endothelial cells, We have previously shown that a nonadher ent yolk sac cell population (WGA(+), density <1.077, AA4.1(+)) can gi ve rise to B cells, T cells, and myeloid cells both in vitro and in vi vo. We now report on the ability of a yolk sac-derived cloned endothel ial cell line (C166) to provide a suitable microenvironment for expans ion of these early precursor cells, Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded > 100 fold within 8 days b y coculture with irradiated C166 cells, Colony-forming ability was ret ained for at least three passages in vitro, with retention of the abil ity to differentiate into T-cell, B-cell, and myeloid lineages, Stem c ell properties were maintained by a significant fraction of nonadheren t cells in the third passage, although these stem cells expressed a se men-hat more mature cell surface phenotype than the initial yolk sac s tem cells, When reintroduced into adult allogeneic immunocompromised ( scid) hosts, they were able to give rise to all of the leukocyte linea ges, including T cells, B cells, and myeloid cells, We conclude that y olk sac endothelial cells can support the stable proliferation of mult ipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent deve lopment and differentiation, for in vivo hematopoietic restitution, an d fur potential use as a vehicle for gene transfer.