Method for the preparation of embryoid bodies (EBs) and uses thereof
Provided are means and methods for producing embryoid bodies (EBs) from multi- or pluripotent cells. In particular, a method of generating embryoid bodies (EBs) is described comprising agitation of a liquid suspension culture of multi- or pluripotent cells in a container until generation of cell aggregates, optionally diluting the suspension, and further agitation of the suspension until formation of EBs. Furthermore, the present invention relates to the use of the novel culturing method and EBs obtained thereby for a variety of applications including genomics, diagnostic assays, teratogenic/embryotoxicological and pharmacological assays as well as for the provision of tissue grafts.
1. A method for producing embryoid bodies (EBs) from pluripotent cells comprising
(i) obtaining a liquid single cell suspension culture of pluripotent cells;
(ii) collecting and suspending the cells in a container to a density of about 0.5×10 6 to 5×10 6 cells/ml;
(iii) rocking the container containing the liquid single cell suspension culture thereby generating cell aggregates; and
(iv) diluting the suspension, and further rocking a container containing the suspension until formation of EBs;
wherein the final concentration of EBs in the suspension culture is about 500 EBs/ml;
wherein the pluripotent cells are selected from the group consisting of embryonic stern (ES) cells, primordial germ (EG) cells and non-embryonic pluripotent stem cells.
2. The method of claim 1 , wherein prior to step (i) the cells are cultured on embryonic mouse fibroblasts (feeder cells).
3. The method of claim 1 or 2 , wherein said pluripotent cells are embryonic stem (ES) cells.
4. The method of claim 3 , wherein said cells are obtained from a, murine ES cell line.
5. The method of claim 1 , wherein the culture medium in any or all of the steps is Iscove's Modified Dulbecco's Media (IMDM) and 20% FCS.
6. The method of claim 1 , wherein the culture conditions in any or all of steps (i) through (iii) comprise 37° C., 5% CO 2 and 95% humidity.
7. The method of claim 1 , wherein said culture of pluripotent cells has a concentration of about 1×10 6 to 5×10 6 cells/ml.
8. The method of claim 1 , wherein the suspension in step (iii) is cultured for about 6 hours.
9. The method of claim 1 , wherein the suspension is cultured for about 16 to 20 hours.
10. The method of any one of claims 1 , 8 and 9 , wherein the suspension in step (iv) is cultured in T25 flasks.
11. The method of claim 1 , wherein said dilution in step (iv) is 1:10.
12. The method of claim 1 , further comprising diluting the cell aggregates to the desired final concentration.
13. The method of claim 1 , further comprising culturing the EBs under conditions allowing differentiation of the EBs into cardiomyocytes.
14. The method of claim 13 , further comprising selection of cardiomyocytes by use of one or more selectable markers or agents or both.
15. The method of claim 14 , wherein said cell is genetically engineered.
16. The method of any one of claims 14 and 15 , wherein said cell comprises a selectable marker or a reporter gene or both.
17. The method of claim 16 , wherein said cell comprises a selectable marker gene operably linked to a cell type-specific regulatory sequence.
18. The method of claim 17 , wherein said selectable marker confers resistance to puromycin.
19. The method of claim 17 , wherein said cell type-specific regulatory sequence is atrial- or ventricular-specific.
20. The method of claim 19 , wherein said regulatory sequence is a cardiac-specific regulatory sequence selected from the group consisting of the promoters of alpha-myosin heavy charin (alpha-MHC) and ventricular myosin light chain 2 (MLC2v).
21. The method of claim 16 , wherein said cell comprises a reporter gene operably linked to a cell type-specific regulatory sequence.
22. The method of claim 21 , wherein said cell type-specific regulatory sequence of the reporter gene is the same as said cell type-specific regulatory sequence of the marker gene.
23. The method of claim 22 , wherein said reporter is enhanced green fluorescent protein (EGFP).
24. The method of claim 21 , wherein said cell type-specific regulatory sequence is atrial- or ventricular-specific.
25. The method of claim 16 , wherein said marker gene and said reporter gene are contained on the same recombinant nucleic acid molecule.
26. The method of claim 25 , wherein said marker gene and said reporter gene are contained on the same cistron.
27. A method for producing embryoid bodies (EBs) from pluripotent cells comprising
(i) obtaining a liquid single cell suspension culture of pluripotent cells;
(ii) collecting and suspending the cells in a container to a density of about 0.1×10 6 to 1×10 6 cells/ml;
(iii) rocking the container containing the liquid single cell suspension culture thereby generating cell aggregates; and
(iv) rocking the container containing the suspension until formation of EBs;
wherein the pluripotent cells are selected from the group consisting of embryonic stem (ES) cells, primordial germ (EG) cells and non-embryonic pluripotent stem cells, and wherein a 10 ml aliquot of a suspension in (ii) comprising 0.2×10 6 pluripotent cells yields sufficient EBs to seed six 20 ml suspensions each comprising 1000 EBs.
28. The method of claim 27 , wherein prior to step (iii) the cells are cultured on embryonic mouse fibroblasts (feeder cells).
29. The method of claim 27 or 28 , wherein said pluripotent cells are embryonic stem (ES) cells.
30. The method of claim 29 , wherein said cells are obtained from a murine ES cell line.
31. The method of claim 27 , wherein the culture medium in any or all of the steps is Iscove's Modified Dulbecco's Media (IMDM) and 20% FCS.
32. The method of claim 27 , wherein the culture conditions in any or all of steps (i) through (iv) comprise 37° C., 5% CO 2 and 95% humidity.
33. The method of claim 27 , wherein said culture of pluripotent cells has a concentration of about 0.1×10 6 to 0.5×10 6 cells/ml.
34. The method of claim 27 , wherein the suspension is cultured for about 48 hours.
35. The method of claim 27 , further comprising culturing the EBs under conditions allowing differentiation of the EBs into cardiomyocytes.
36. The method of claim 35 , further comprising selection of cardiomyocytes by use or one or more selectable markers or agents or both.
37. The method of claim 36 , wherein said cell is genetically engineered.
38. The method of claim 36 or 37 , wherein said cells comprises a selectable marker or a reporter gene or both.
39. The method of claim 38 , wherein said cell comprises a selectable marker gene operably linked to a cell type-specific regulatory sequence.
40. The method of claim 39 , wherein said selectable marker confers resistance to puromycin.
41. The method of claim 39 , wherein said cell type-specific regulatory sequence is atrial- or ventricular-specific.
42. The method of claim 41 , wherein said regulatory sequence is a cardiac-specific regulatory sequence selected from the group consisting of the promoters of alpha-myosin heavy chain (alpha-MHC) and ventricular myosin light chain 2 (MLC2v).
43. The method of claim 38 , wherein said cell comprises a reporter gene operably linked to a cell type-specific regulatory sequence.
44. The method of claim 43 , wherein said cell type-specific regulatory sequence of the reporter gene is the same as said cell type-specific regulatory sequence of the marker gene.
45. The method of claim 44 , wherein said reporter is enhanced green fluorescent protein (EGFP).
46. The method of claim 43 , wherein said cell type-specific regulatory sequence is atrial- or ventricular-specific.
47. The method of claim 38 , wherein said marker gene and said reporter gene are contained on the same recombinant nucleic acid molecule.
48. The method of claim 37 , wherein said marker gene and said reporter gene are contained on the same cistron.