Human embryonic stem cells and culturing methods thereof

• US 20060030040A1
• Filed: 09/23/2005
• Published: 02/09/2006
• Est. Priority Date: 12/09/2003
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Chen, Wann-Hsin, Hsu, Lih-Tao, Yang, Mei-Ju, Kuo, Chao-Ying, Huang, Jun-Jae, Chang, Kuang-Ning
• Granted Patent: US 7,682,826 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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

Provided herein are human embryonic stem cells and their method of culturing. In one embodiment, the human embryonic stem cells are maintained in an environment containing an extracellular matrix isolated from feeder cells and a conditioned medium pre-conditioned by the feeder cells; the feeder cells are pre-inactivated by either gamma ray radiation or by mitomycin C treatment. The cultured human embryonic stem cells remain substantially undifferentiated and maintain their pluripotency to differentiate into three germ layer cells.

Claims

1. A culture system for maintaining the undifferentiated growth of human embryonic stem cells comprising:

a substrate covered with an extracellular matrix isolated from feeder cells;

a conditioned medium that was preconditioned by the feeder cells, and undifferentiated human embryonic stem cells;

wherein the feeder cells are pre-inactivated by gamma ray irradiation or by treatment with mitomycin C.

2. The culture system of claim 1, wherein at least 60% of the human embryonic stem cells remain substantially undifferentiated after 5 passages.

3. The culture system of claim 2, wherein at least 78% of the human embryonic stem cells remain substantially undifferentiated after 5 passages.

4. The culture system of claim 1, wherein the feeder cells are selected from the group consisting of human foreskin fibroblasts (HFF), primary mouse embryonic fibroblasts (PMEF), a mouse embryonic fibroblast cell line (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human fetal muscle (HFM), human fetal skin cells (HFS), human adult skin cells, human adult fallopian tubal epithelial cells (HAFT) or human marrow stromal cells (hMSCs).

5. The culture system of claim 4, wherein the feeder cells is mouse embryonic fibroblasts or human foreskin fibroblasts.

6. The culture system of claim 1, wherein the extracellular matrix is prepared by culturing the feeder cells, lysing the feeder cells with alkali solution or trinitrotoluene, and then washing what remains after lysing.

7. The culture system of claim 1, wherein the conditioned medium is prepared by the steps of:

(a) inactivating the feeder cells by gamma ray irradiation or by treatment with mitomycin C;

(b) culturing the cells obtained in step (a) in a culture medium for at least 1 day; and

(c) collecting the culture medium of step (b) as the conditioned medium.

8. The culture system of claim 7, wherein the concentration of mitomycin C is between about 5 μ

g/ml to about 25 μ

g/ml.

9. The culture system of claim 8, wherein the concentration of mitomycin C is about 10 μ

g/ml.

10. The culture system of claim 7, wherein the dosage range of gamma ray is between about 3,000 rads to about 6,000 rads.

11. A method of proliferating human embryonic stem cells comprising culturing the stem cells in an environment containing an extracellular matrix isolated from feeder cells, and a conditioned medium;

wherein the feeder cells are pre-inactivated by gamma ray irradiation or by treatment with mitomycin C.

12. The method of claim 11, wherein at least 60% of the human embryonic stem cells remain substantially undifferentiated after 5 passages.

13. The method of claim 12, wherein at least 78% of the human embryonic stem cells remain substantially undifferentiated after 5 passages.

14. The method of claim 11, wherein the feeder cells are selected from the group consisting of human foreskin fibroblasts (HFF), primary mouse embryonic fibroblasts (PMEF), a mouse embryonic fibroblast cell line (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human fetal muscle (HFM), human fetal skin cells (HFS), human adult skin cells, human adult fallopian tubal epithelial cells (HAFT) or human marrow stromal cells (hMSCs).

15. The method of claim 14, wherein the feeder cells is human foreskin fibroblasts or mouse embryonic fibroblasts.

16. The method of claim 11, wherein the extracellular matrix is prepared by culturing the feeder cells, lysing the feeder cells with alkali solution or trinitrotoluene, and then washing what remains after lysing.

17. The method of claim 11, wherein the conditioned medium is prepared by the steps of:

(a) inactivating the feeder cells by gamma ray irradiation or treatment with mitomycin C;

(b) culturing the cells obtained in step (a) in a culture medium for at least 1 day; and

(c) collecting the culture medium of step (b) as the conditioned medium.

18. The method of claim 17, wherein the concentration of mitomycin C is between about 5 μ

g/ml to about 25 μ

/ml.

19. The method of claim 18, wherein the concentration of mitomycin C is about 10 μ

/ml.

20. The method of claim 17, wherein the dosage range of gamma ray is between about 3,000 rads to about 6,000 rads.

21. An isolated, population of pluripotent human embryonic stem cells obtained by the method of claim 11.

22. A cryopreserved human embryonic stem cells prepared by the method of claim 11.