Methods of expanding embryonic stem cells in a suspension culture

• US 9,834,749 B2
• Filed: 05/25/2015
• Issued: 12/05/2017
• Est. Priority Date: 08/02/2006
• Status: Active Grant

• 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): EPPS -SMITH, JANET L
• Application Number:
  US14/720,853
• Publication Number:
  US 20150252326A1
• Time in Patent Office: 925 Days
• Field of Search: 435366, 435375, 435325
• 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 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2533/12 Glass
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

A method of expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing the ESCs in a suspension culture under culturing conditions devoid of substrate adherence is provided. Also provided are a method of deriving ESC lines in the suspension culture and methods of generating lineage-specific cells from ESCs which were expanded in the suspension culture of the present invention.

Claims

1. A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture without substrate adherence under culturing conditions which allow expansion of the pluripotent stem cells in the undifferentiated state for at least 5 passages, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.

2. The method of claim 1, wherein said culturing conditions comprise culturing the human pluripotent stem cells in a culture vessel having an internal surface designed to prevent attachment or adherence of said pluripotent stem cells to said surface.

3. The method of claim 1, wherein said substrate comprises components of extracellular matrix, a glass microcarrier or beads.

4. The method of claim 1, wherein said culturing conditions comprise a serum-free culture medium selected from the group consisting of:

a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), a culture medium comprising an IL6RIL6 chimera and a culture medium which comprises a TGFb isoform, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.

5. A method of generating lineage-specific cells from human pluripotent stem cells, the method comprising:

(a) culturing the human pluripotent c stem cells in a suspension culture according to the method of claim 1, to thereby obtain expanded, undifferentiated human embryonic stem cells; and

(b) subjecting said expanded, undifferentiated human embryonic stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells;

thereby generating the lineage-specific cells from the human embryonic stem cells.

6. A method of generating embryoid bodies from human pluripotent stem cells, the method comprising:

(a) culturing the human pluripotent stem cells in a suspension culture according to the method of claim 1, to thereby obtain expanded, undifferentiated human pluripotent stem cells; and

(b) subjecting said expanded, undifferentiated human pluripotent stem cells to culturing conditions suitable for differentiating said human embryonic stem cells to embryoid bodies;

thereby generating the embryoid bodies from the human pluripotent stem cells.

7. A method of generating lineage-specific cells from pluripotent stem cells, the method comprising:

(a) culturing the human pluripotent stem cells in a suspension culture according to the method of claim 1, to thereby obtain expanded, undifferentiated human pluripotent stem cells;

(b) subjecting said expanded, undifferentiated human pluripotent stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated human pluripotent stem cells to embryoid bodies; and

(c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells;

thereby generating the lineage-specific cells from the human pluripotent stem cells.

8. The method of claim 4, wherein said TGFβ

isoform is a TGFβ

isoform 1 (TGFβ

1).

9. The method of claim 4, wherein said TGFβ

isoform is a TGFβ

isoform 3 (TGFβ

3).

10. The method of claim 4, wherein said sIL6R is present at a concentration of 15-30 ng/ml.

11. The method of claim 4, wherein said LIF is present at a concentration of at least 2000 units per milliliter (u/ml).

12. The method of claim 1, wherein said culturing is effected under xeno-free conditions.

13. The method of claim 8, wherein said TGFβ

1 is present at a concentration of at least 0.06 ng/ml.

14. The method of claim 9, wherein said TGFβ

3 is present at a concentration of or at least 0.5 ng/ml.

15. The method of claim 1, wherein said culture conditions further comprise basic fibroblast growth factor (bFGF), and optionally wherein said bFGF is at a concentration of at least 2 ng/ml.

16. The method of claim 1, wherein said pluripotent stem cells comprise embryonic stem cells.

17. The method of claim 1, wherein said pluripotent stem cells remain in the undifferentiated state without adherence to said substrate for at least 5 passages.

18. The method of claim 1, wherein said pluripotent stem cells are capable of forming teratomas containing representative tissues of all three embryonic germ layers.

19. The method of claim 1, wherein said pluripotent stem cells express OCT4.