ABSTRACT
Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast.
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Hematopoiesis/genetics , Proto-Oncogene Proteins , Stem Cells/drug effects , Transcription Factors , Activins , Basic Helix-Loop-Helix Transcription Factors , Cell Count , Cell Differentiation , Cell Line , DNA-Binding Proteins/genetics , Flow Cytometry , Gene Expression Regulation, Developmental , Gene Targeting , Humans , Inhibins/pharmacology , Mesoderm/metabolism , Mutation , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Receptors, Growth Factor/genetics , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Signal Transduction , Stem Cells/physiology , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.
