Development of Pluripotential Embryonic Stem Cells from Rat Blastocyst

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To establish pluripotential diploid cells from rat embryos, we cultured blastocysts in medium containing mouse leukemia inhibiting factor (LIF) and insulin-like growth factor (IGF) -II.Combination of mouse LIF (5,000 units/ml) and rat IGF-II (100ng/ml) promoted the growth of inner cell mass (ICM) of rat blastocyst. The form of the cells derived from ICM was similar to that of mouse embryonic stem (ES) cell. We established 9 and 3 lines on ACI and a hybrid (ACI * Wistar-Imamichi) rat, respectively. For evaluating in vivo pluripotency of the rat ES-like cells, 8-cell embryos of Wistar-Imamichi were co-cultured with the established cells to make chimeric rats. On the cell lines derived from ACI rat, 25 neonates were obtained from 349 chimeric embryos that implanted into pseudopregnancy rat. On the neonates, no chimeric rat was observed. By microinjection method, chimeric embryos were produced between the established cells and blastocysts of Wistar-Imamichi. On the cell lines derived from both ACI and the hyberid rats, 304 chimeric embryos were produced. Chimeric rats were not, however, founded in 206 neonates. These findings suggest that, in addition to LIF and IGF-II,other factor may be needed to establish pluripotential ES cells from rat embryos.