Medium and culture of embryonic stem cells

A medium comprising salts, vitamins, amino acids, glucose, albumin, minerals, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, a fibroblast growth factor, and at least one member selected from gamma-aminobutyric acid, pipecolic acid, and lithium, in sufficient amounts to maintain stem cells grown in the medium in an undifferentiated state through multiple culture passages.

A method for initiating a cultured line of primate pluripotent stem cells without the use of feeder cells or conditioned medium, the method comprising the step of:

• plating cells from a blastocyst onto a matrix in a medium including salts, vitamins, amino acids, glucose, albumin, minerals, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, a fibroblast growth factor and at least one member selected from gamma-aminobutyric acid, pipecolic acid, and lithium, in sufficient amounts to originate and maintain a new proliferating stem cell line in an undifferentiated state.

A method of increasing cloning efficiency of primate pluripotent stem cells comprising:

• culturing the primate pluripotent stem cells on a matrix in a medium without feeder cells or conditioned media, the medium comprising salts, vitamins, amino acids, glucose, albumin, minerals, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, a fibroblast growth factor, less than about 0.1 mM beta-mercaptoethanol and at least one member selected from gamma-aminobutyric acid, pipecolic acid, and lithium in sufficient amounts to maintain the cells in an undifferentiated state through multiple successive culture passages and such that the cloning efficiency of the pluripotent cells is increased by at least 10% compared to pluripotent stem cells cultured in the same medium, except wherein the beta-mercaptoethanol concentration is higher than about 0.1 mM.

A method of increasing cloning efficiency of primate pluripotent stem cells without the need for feeder cells or conditioned medium, the method comprising the step of:

• culturing the primate pluripotent stem cells on a matrix that comprises a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma or a matrix that comprises human matrix proteins collagen IV, fibronectin, laminin, and vitronectin in a medium without feeder cells or conditioned media, the medium comprising salts, vitamins, amino acids, glucose, albumin, minerals, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, a fibroblast growth factor, less than about 0.1 mM beta-mercaptoethanol, gamma-aminobutyric acid, pipecolic acid, lithium, and transforming growth factor beta, in sufficient amounts to maintain the human stem cells in an undifferentiated state, wherein at least 90% of the cells in culture are positive for the transcription factor Oct-4 through multiple successive culture passages and wherein the cloning efficiency of the pluripotent cells is increased by at least 10% compared to pluripotent stem cells cultured in the same medium, except wherein the beta-mercaptoethanol concentration is higher than about 0.1 mM.

A medium comprising salts, vitamins, amino acids, glucose, albumin, minerals, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, a fibroblast growth factor, less than about 0.1 mM beta-mercaptoethanol, and at least one member selected from gamma-aminobutyric acid, pipecolic acid, and lithium, in sufficient amounts to maintain stem cells grown in the medium in an undifferentiated state through multiple culture passages.