Maintenance of undifferentiated mouse ES cells in suspension by the serum and feeder-free defined culture condition

Mouse embryonic stem (ES) cells are indispensable for gene targeting approaches to study gene functions and regulations, the production of animal models for human diseases, and in vitro cell differentiation studies. The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium is free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions and activation of the WNT signaling pathway. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. Keywords: reference design,replicate design RNA was extracted by using Isogen (Wako) according to the manufacturer's instructions. First-strand cDNA was prepared by using Super Script reverse transcriptase (Invitrogen) primed with random hexamer in a 20ml reaction mixture containing 5 mg of total RNA. A total of 0.5 ml of the first-strand cDNA mixture was used for PCR with Taq polymerase (TAKARA) performed in a 50-ml volume. The following conditions were used: 10 min at 95C followed by 40 cycles of 15 seconds at 95C and 1 min at 60C (for Oct4, Nanog, ERas, Rex-1, Snail1, Gata4, Slug, and Gapdh) or 2 min at 94C followed by 25 cycles of 30 seconds at 94C, 30 seconds at 56C, and 1 min at 72C (for Itgb8, Cdh11, and Col3a1). The PCR reaction products were separated by gel electrophoresis, and the DNA bands were visualized under ultraviolet light for photography.