Methods of expanding embryonic stem cells in a suspension culture

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 (TGFβ1).

9. The method of claim 4 , wherein said TGFβ isoform is a TGFβ isoform 3 (TGFβ3).

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.