Introduction: Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both\r\ntransplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional\r\nhepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical\r\napplications.\r\nMethods: In this study, we investigated whether a large number of functional hepatocytes can be differentiated\r\nfrom mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating\r\nbioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then\r\ndifferentiated into hepatocytes.\r\nResults: During the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of\r\nnormal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level\r\nin the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the\r\nEBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of\r\nhepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor\r\nexhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density\r\nlipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the\r\nEB-derived cells from day-14 EBs and the cells� culture supernatant were injected into nude mice, the transplanted\r\ncells were engrafted into the recipient livers.\r\nConclusions: Large quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor\r\nvia EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation\r\nand the development of bioartificial livers.
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