ALBERT

Beschreibung: Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular and molecular mechanisms regulating germ layer fate determination and tissue formation. Both of hemangioblasts and cardiomyocytes are thought to be derived from lateral plate mesoderm of early embryo, but little is known about the phenotypic specification in these two lines of differentiation at the divergent point through development. Utilizing the in vitro differentiation system of murine ES cells in the presence or absence of stromal cell layer, and gene expression data in the microarray analysis, we determined a set of cell-surface molecules as specific markers, which enabled efficient enrichment of hemangioblasts or cardiac myocytes. Furthermore, we represent the evidences that the sorted fraction with hemangioblast-specific markers markedly produced both of blood island and endotherial colonies in stromal-free culture system. Materials and Methods Murine embryonic stem cell line, EB5 was cultured on gelatin-coated plates for mesodermal differentiation after withdrawal of LIF and Blastcidin. The expression of surface molecules related to mesodermal differentiation was evaluated with flowcytometry or quantitative RT-PCR techniques. The cell population defined by combination of the candidate surface molecules was sorted at day 5 and seeded on PA-6 or OP-9 stromal cell layers, or in conditioned medium of PA-6 cells for stroma-free assay until day 12 of differentiation. Differentiational direction of each culture sample was evaluated by the expression of genes or membrane molecules specific for tissue lineages with quantitative RT-PCR or flowcytometric techniques. The cardiogenic potential was also assessed by scoring beating colonies co-cultured on stromal cells. Results and Discussion In combination with cell surface expression of Flk-1 (VEGFR2), which is known to be a restricted marker for mesodermal tissues during early embryonic development, upregulated expression of Platelet-Derived Growth Factor (PDGF) receptor alpha on cell surface was detected more frequently in the fraction containing pre-cardiomyocytic cells. In contrast, negative fraction for PDGFRalpha contributed to efficient formation of hematopoietic burst colonies growing together with endotherial cells engulfing DiI- acetylated LDL. We also cofirmed that these suspended hematopoietic cells were spurting out of hemangioblast-like compartment in time-lapse imaging analysis. Our findings may provide a new experimantal window to analyze the common mesodermal precursors for hemangioblasts and cardiomyocytes in vitro. We will search for tissue stem cells bearing the corresponding surface markers in human cell sources, and evaluate their differentiation potentials and therapeutic effect on ischemic diseases.