Primate pluripotent stem cells cultured in medium containing gamma-aminobutyric acid, pipecolic acid and lithium

• US 8,158,424 B2
• Filed: 04/05/2010
• Issued: 04/17/2012
• 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:
  US12/754,574
• Publication Number:
  US 20100304481A1
• Time in Patent Office: 743 Days
• Field of Search: None
• US Class Current: 435/377
• 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 method for culturing primate pluripotent stem cells in an undifferentiated state on a matrix without the need for feeder cells or conditioned medium, the method comprising the step of:

culturing the primate pluripotent stem cells on an extracellular 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, 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.

2. The method of claim 1 wherein the matrix comprises a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma or a matrix that comprises human matrix proteins collagen IV and at least one member selected from fibronectin, laminin, and vitronectin.

3. The method of claim 1 wherein the matrix comprises collagen IV, fibronectin, laminin, and vitronectin.

4. The method of claim 1 wherein the medium comprises gamma amino butyric acid, pipecolic acid, lithium, and optionally transforming growth factor beta.

5. The method of claim 1 wherein when the matrix 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, and the medium comprises 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, 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.

6. An in vitro cell culture comprising in a culture vessel:

primate pluripotent stem cells;

an extracellular matrix, on which the stem cells can grow; and

a culture medium, wherein the medium comprises 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 the human stem cells in an undifferentiated state through multiple culture passages, the medium being free of feeder cells and never having been exposed to feeder cells.

7. The cell culture of claim 6, wherein when the matrix comprises human matrix proteins collagen IV, fibronectin, laminin, and vitronectin, and the medium comprises 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, 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.

8. The cell culture of claim 6, wherein the medium is free of products from non-human animals.

9. The cell culture of claim 6 wherein the cells are human pluripotent stem cells.

10. The cell culture of claim 6, wherein the medium is capable of maintaining the cells through over twenty passages in culture while the cells remain undifferentiated, maintain pluripotency and maintain stable karyotype.

18. The cell culture of claim 9 wherein the cells do not exhibit the sialic acid residue Neu5Gc.

11. 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.

12. 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 an extracellular 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.

13. The method of claim 12 wherein the matrix comprises collagen and at least one of fibronectin, vitronectin and laminin.

14. The method of claim 12 wherein the medium comprises at least two members of gamma amino butyric acid, pipecolic acid, and lithium, and optionally transforming growth factor beta.

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

culturing the primate pluripotent stem cells on an extracellular 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.

16. 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.

17. 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.