Production of medaka chimeras from in vitro cultured embryonic cells

Abstract

In order to develop an experimental technology employing fish embryonic stem (ES) cells, we attempted to culture cells dissociated from blastula-stage embryos of the medaka (Oryzias latipes). Three types of culture conditions were tested with regard to the culture medium, coating of the culture dishes or addition of conditioned medium. Cells incubated for 1 day under each set of culture conditions were transplanted into recipient embryos to examine whether they retained the ability to form chimeric fish, which was judged according to the body pigmentation. In a control experiment in which intact blastomeres were transplanted, chimeras were produced at a frequency of 32% among survivors. When isolated embryonic cells were dissociated and incubated in a culture medium, most of the cells did not attach to the culture dishes. The frequency of chimera formation by these cells was 15%. Use of gelatin + poly-lysine-coated dishes improved the cell attachment and the cells formed aggregated colonies, but the frequency of chimera formation by these cells decreased to 7%. Further addition of conditioned medium enhanced the cell attachment, and caused the cells to spread individually over the dishes. However, the ability of the cells to form chimeras was lost following this treatment. The results of the present study demonstrate that it is possible to generate viable chimeras from cultured embryonic cells, although further improvement of the culture conditions is necessary to develop a method for culturing fish ES cells.