GENERATION OF HEMATOPOIETIC COLONY-FORMING CELLS FROM EMBRYONIC STEM-CELLS - SYNERGY BETWEEN A SOLUBLE FACTOR FROM NIH-3T3 CELLS AND HEMATOPOIETIC GROWTH-FACTORS

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs, With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitory factor were cultured under different conditions, Low numbers of day-g EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-l, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture, When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells, Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-g EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells. (C) 1995 by The American Society of Hematology.