Culture medium containing gamma-aminobutyric acid, pipecolic acid or lithium for the maintenance of stem cells in an undifferentiated state

• US 8,426,203 B2
• Filed: 03/22/2012
• Issued: 04/23/2013
• Est. Priority Date: 09/08/2004
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A., Ludwig, Tenneille
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/427,548
• Publication Number:
  US 20120178161A1
• Time in Patent Office: 397 Days
• Field of Search: None
• US Class Current: 435/404
• CPC Class Codes: C12N 2500/12 Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/50 Soluble polymers, e.g. polyethyleneglycol [PEG]
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/845 Gamma amino butyric acid [GABA]
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Previous methods for culturing primate pluripotent stem cells have required either fibroblast feeder cells or a medium which was exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. It has now been found that high levels of fibroblast growth factor in a medium together with at least one of gamma aminobutyric acid, pipecolic acid, and lithium, enables pluripotent stem cells to remain undifferentiated indefinitely through multiple passages, even without feeder cells or conditioned medium. Without beta-mercaptoethanol, the medium improves cloning efficiency. Also, a matrix of human proteins can be used to culture the undifferentiated cells without exposing the cells to animal products. Further disclosed are new primate pluripotent cell lines made using the defined culture conditions, including the medium and the matrix. Such new cell lines will have never been exposed to animal cells, animal products, feeder cells or conditioned medium.

Claims

1. 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, and optionally transforming growth factor beta, in sufficient amounts to maintain stem cells grown in the medium in an undifferentiated state through multiple culture passages.

4. The medium of claim 1, 2 or 3, wherein the insulin and transferrin are recombinant proteins.

5. The medium of claim 1, 2 or 3, wherein the stem cells are primate pluripotent stem cells.

2. 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 two members selected from gamma-aminobutyric acid, pipecolic acid and lithium, and optionally transforming growth factor beta, in sufficient amounts to maintain stem cells grown in the medium in an undifferentiated state through multiple culture passages.

3. 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, gamma-aminobutyric acid, pipecolic acid and lithium, and optionally transforming growth factor beta, in sufficient amounts to maintain stem cells grown in the medium in an undifferentiated state through multiple culture passages.