a study on isolation and clonal expansion of the stem cell of hepatic stellate cells.

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A stem cell of the hepatic stellate cells was established from mouse embryos. We observed morphologically the process of liver bud formation mixing mesenchymal tissue and foregut in the mouse embryos. During 10-14 somite stage, the hepatic diverticulum was formed and during 18-25 somite stage the foregut entered the septum transversum and the liver bud formation was started. Cell masses obtained from the liver bud containing the hepatic diverticulum and the septum transversum were quickly removed and treated with dispase to get the suspension of isolated cells. The isolated cells were cultured in (1)polystyrene culture dishes, (2)gelatin-coated polystyrene dishes, or (3)polystyrene dishes with a feeder layer of mouse embryo fibroblasts. Three culture media were used : (1)DMEM high glucose medium containing 10% calf serum, (2)DMEM/F12 medium containing 15% calf serum, and (3)ES cell growing medium containing 15% calf serum. To inhibit cell differentiation, a leukemia inhibiting factor (1,000 u/ml) was added to each medium. The best condition was obtaining with ES cell growing medium in gelatin-coated polystyrene dishes. After getting colonies in the dish, each colony was cloned. Cell type was identified by immunofluorescence by using specific antibody against BMP4, AFP, or FLT1(VEGFR), and each antibody demonstrated stellate cells, parenchymal cells, and endothelial cells, respectively. Thus, we established the stem cell of the hepatic stellate cells. The same clone was obtained from flt1 knock out mouse embryos.