Provided are novel serum-free culture media which comprise basic fibroblast growth factor (bFGF), transforming growth factor beta-3 and ascorbic acid at a concentration of at least about 50 microgram/ml； ascorbic acid at a concentration range of about 400-600 microgram/ml, bFGF at a concentration range of about 50-200 ng/ml, xeno-free serum replacement and a lipid mixture； the IL6RIL6 chimera at a concentration range of about 50-200 picogram per milliliter (pg/ml)； or leukemia inhibitory factor (LIF) at a concentration of at least 2000 units/ml； wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state in the absence of feeder cell support. Also provided are cell cultures comprising pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem (iPS) cells and the novel culture media, methods of using same for expanding pluripotent stem cells in an undifferentiated state using two-dimensional or three-dimensional culture systems； and methods of expanding iPS cells in a suspension culture devoid of substrate adherence and cell encapsulation.
