Culturing human embryonic stem cells in medium containing pipecholic acid and gamma amino butyric acid

• US 7,442,548 B2
• Filed: 09/08/2005
• Issued: 10/28/2008
• 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:
  US11/221,516
• Publication Number:
  US 20070238170A1
• Time in Patent Office: 1,146 Days
• Field of Search: 435/366, 435/377, 435/384
• 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 2500/33 other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/845 Gamma amino butyric acid [GABA]
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• 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

Abstract

Previous methods for culturing human embryonic stem cells have required either fibroblast feeder cells or a medium which has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. It has now been found that if high levels of fibroblast growth factor are used in a medium with gamma amino butyric acid, pipecholic acid, lithium and lipids, the stem cells will remain undifferentiated indefinitely through multiple passages, even without feeder cells or conditioned medium. A humanized matrix of human proteins can be used as a basement matrix to culture the cells. New lines of human embryonic stem cells made using these culture conditions, the medium and the matrix, will never have been exposed to animal cells, animal products, feeder cells or conditioned medium.

Claims

1. A method for initiating a new cultured line of human embryonic 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 albumin, minerals, vitamins, amino acids, glucose, a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium, lipids, a transferrin or a transferrin substitute and insulin or an insulin substitute in sufficient amounts to originate and maintain a new proliferating stem cell line in an undifferentiated state.

2. The method of claim 1 wherein the medium includes the fibroblast growth factor in a concentration of at least 40 ng/ml.

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

human embryonic stem cells;

a culture medium, the culture medium comprising albumin, minerals, salts, vitamins, amino acids, glucose, a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium, lipids, a transferrin or a transferrin substitute and insulin or an insulin substitute in sufficient amounts to maintain the 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; and

a humanized matrix made from human collagen and at least two of human proteins selected from fibronectin, vitronectin and laminin.

4. The culture of claim 3 wherein the matrix includes all of collagen, fibronectin, vitronectin and laminin.

5. The culture of claim 3 wherein the medium includes the fibroblast growth factor in a concentration of at least 40 ng/ml.

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

human embryonic stem cells;

a humanized matrix made from human collagen and at least two of the human proteins selected from fibronectin, vitronectin and laminin; and

a culture medium, the culture medium comprising albumin, minerals, vitamins, amino acids, glucose, a fibroblast growth factor, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, and at least two members selected from gamma amino butyric acid, pipecholic acid, and lithium in sufficient amounts to maintain the 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. A method of culturing new human embryonic stem cells comprising the steps of

(a) isolating the inner cell mass of an embryo in the blastocyst stage;

(b) culturing the cells from step (a) in a medium including albumin, minerals, vitamins, amino acids, glucose, a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium, lipids, a transferrin or a transferrin substitute and insulin or an insulin substitute in sufficient amounts to maintain the stem cells in an undifferentiated state through multiple culture passages, the culturing step being conducted on a matrix of human proteins comprising at least three of the proteins selected from collagen, fibronectin, vitronectin, and laminin; and

(c) serially expanding the new cells which proliferate on the medium.

8. A culture of cells comprising human embryonic stem cells growing on a matrix of human proteins that comprises at least three of the proteins selected from collagen, fibronectin, vitronectin, and laminin, the stem cells from a lineage which has never been exposed to animal cells, animal proteins, feeder cells or conditioned medium, the cell culture medium comprising albumin, minerals, vitamins, amino acids, glucose, a fibroblast growth factor, gamma amino butyric acid, pipecholic acid, lithium, lipids, a transferrin or a transferrin substitute, and insulin or an insulin substitute capable of maintaining the cells through over twenty passages in culture while the cells remain undifferentiated, maintain pluripotency and maintain normal karyotype.

9. A culture of cells comprising human embryonic stem cells which do not exhibit the sialic acid Neu5Gc, wherein the culture comprises (i) human embryonic stem cells on a matrix of human proteins that comprises at least three of collagen, fibronectin, vitronectin, or lamiin and (ii) a cell culture medium that comprises albumin, minerals, vitamins, amino acids, glucose, a fibroblast growth factor, lipids, a transferrin or a transferrin substitute, insulin or an insulin substitute, and at least two members selected from gamma amino butyric acid, pipecholic acid, and lithium.