Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor

• US 10,066,205 B2
• Filed: 07/04/2016
• Issued: 09/04/2018
• Est. Priority Date: 12/16/2002
• Status: Expired due to Term

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/201,537
• Publication Number:
  US 20160312180A1
• Time in Patent Office: 792 Days
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/84 from mammals
• 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 2501/10 Growth factors
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0031 Serum-free culture media
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0056 Xeno-free medium
• 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 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0611 : Primordial germ cells, e.g. embryonic germ cells [EG]
• CPC Class Codes: C12N 5/0623 : Stem cells
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention is of methods of establishing and propagating human embryonic stem cell lines using feeder cells-free, xeno-free culture systems and stem cells which are capable of being maintained in an undifferentiated, pluripotent and proliferative state in culture which is free of xeno contaminants and feeder cells.

Claims

1. A method of maintaining a human pluripotent stem cell in an undifferentiated state, the method comprising culturing the human pluripotent stem cell in a serum-free culture medium under adherent conditions comprising culturing the human pluripotent stem cell on a matrix comprising basement membrane or extracellular matrix components, said culture medium comprising bFGF, TGFβ

1 and at least one of transferrin and/or insulin, wherein said TGFβ

₁ is provided at a concentration of at least 0.06 ng/ml, wherein said culture medium is capable of maintaining the stem cell in an undifferentiated state, thereby maintaining the stem cell in the undifferentiated state.

2. The method of claim 1, wherein said culture medium comprises bFGF, TGFβ

1, transferrin and insulin.

3. The method of claim 1, wherein said culture medium is free of an animal contaminant.

4. The method of claim 1, wherein said culture medium is free of feeder cell contaminants.

5. The method of claim 1, wherein said bFGF is provided at a concentration of at least 2 ng/ml.

6. The method of claim 1, further comprising leukemia inhibitor factor (LIF).

7. The method of claim 6, wherein said LIF is provided at a concentration of at least 500 units/ml.

8. The method of claim 1, wherein said bFGF is provided at a concentration range of 2-8 ng/ml.

9. The method of claim 1, wherein said TGFβ

1 is provided at a concentration range of 0.06-0.24 ng/ml.

10. The method of claim 1, wherein said TGFβ

₁ is provided at a concentration range of 0.06-0.24 ng/ml, and said bFGF is provided at a concentration of at least 2 ng/ml.

11. The method of claim 6, wherein said LIF is provided at a concentration of 500-2000 units/ml.

12. The method of claim 6, wherein said TGFβ

₁ is provided at a concentration range of 0.06-0.24 ng/ml, said bFGF is provided at a concentration of at least 2 ng/ml and said LIF is provided at a concentration of 500-2000 units/ml.

13. The method of claim 1, further comprising expanding the human pluripotent stem cell in an undifferentiated state for at least 5 passages in said medium.

14. The method of claim 1, wherein said matrix is selected from the group consisting of human plasma fibronectin matrix, recombinant human plasma fibronectin matrix, human cellular fibronectin matrix, recombinant human cellular fibronectin matrix, and synthetic fibronectin.