Monkey-origin embryonic stem cells

• US 20030157710A1
• Filed: 12/13/2002
• Published: 08/21/2003
• Est. Priority Date: 06/15/2000
• Status: Active Grant

• Current Assignee: Tanabe Seiyaku Co Ltd (Mitsubishi Chemical Group Corp.)
• Original Assignee: Tanabe Seiyaku Co Ltd (Mitsubishi Chemical Group Corp.)
• Inventors: Torii, Ryuzo, Hosoi, Yoshihiko, Tada, Takashi, Akuta, Teruo, Nakatsuji, Norio, Iritani, Akira
• Granted Patent: US 7,083,977 B2
• Time in Patent Office: Days
• US Class Current: 435/363
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2503/02 Drug screening
• 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 for producing a monkey-derived embryonic stem cell comprising the steps of carrying out fertilization by insemination by in vitro fertilization or intracytoplasmic sperm injection using a monkey ovum and monkey sperms, thereby giving a fertilized ovum, allowing the fertilized ovum to differentiate into a blastocyst by in vitro culture, and establishing an ES cell line using the blastocyst; the monkey ES cell obtained by the method, a method for screening a reagent for specific differentiation into cell or tissue by using the ES cell; and a differentiated cell or differentiated tissue each differentiated from the ES cell. According to the present invention, applications of the embryonic stem cells to embryological studies clinical applications, experimental models, and the like on primates, studies of diseases, are expected.

Claims

1. A monkey-derived embryonic stem cell obtainable by carrying out a process comprising:

(a) carrying out fertilization by in vitro fertilization or intracytoplasmic sperm injection using a monkey ovum and a monkey sperm, thereby giving a fertilized ovum;

(b) allowing the fertilized ovum obtained in the step (a) to differentiate into a blastocyst by in vitro culture; and

(c) establishing an embryonic stem cell line using the blastocyst obtained in the step (b).

2. The embryonic stem cell according to claim 1, wherein the monkey is Macaca fuscata or Macaca fascicularis.

3. The embryonic stem cell according to claim 1, wherein the monkey is Macaca fascicularis.

4. The embryonic stem cell according to any one of claims 1 to 3, wherein a culture medium selected from the group consisting of TALP solution, TALP-HEPES solution and BWW solution is used in the step (a).

5. The embryonic stem cell according to any one of claims 1 to 4, wherein the in vitro culture in the step (b) is micro-hanging drop culture.

6. The embryonic stem cell according to any one of claims 1 to 5, wherein the in vitro culture in the step (b) is performed using CMRL-1066.

7. The embryonic stem cell according to any one of claims 1 to 6, wherein the temperature for the culture in the step (b) is at 38°

C.

8. The embryonic stem cell according to any one of claims 1 to 7, wherein the conditions for the culture in the step (b) are under conditions at 5% CO₂, 5% O₂ and 90% N₂.

20. A method for screening a reagent for specific differentiation into tissue or cell, characterized by maintaining a cell selected from the group consisting of the monkey-derived embryonic stem cell according to any one of claims 1 to 8 and the cynomologus monkey-derived cell according to any one of claims 17 to 19 in the presence of a test substance.

21. A differentiated cell or differentiated tissue resulting from differentiation from a cell selected from the group consisting of the embryonic stem cell according to any one of claims 1 to 8 and the cynomologus monkey-derived cell according to any one of claims 17 to 19.

9. A method for producing a monkey-derived embryonic stem cell, comprising:

(a) carrying out fertilization by in vitro fertilization or intracytoplasmic sperm injection using a monkey ovum and a monkey sperm, thereby giving a fertilized ovum;

(b) allowing the fertilized ovum obtained in the step (a) to differentiate into a blastocyst by in vitro culture; and

(c) establishing an embryonic stem cell line using the blastocyst obtained in the step (b).

10. The method according to claim 9, wherein the monkey is Macaca fuscata or Macaca fascicularis.

11. The method according to claim 9, wherein the monkey is Macaca fascicularis.

12. The method according to any one of claims 9 to 11, wherein a culture medium selected from the group consisting of TALP solution, TALP-HEPES solution and BWW solution is used in the step (a).

13. The method according to any one of claims 9 to 12, wherein the in vitro culture in the step (b) is a micro-hanging drop culture.

14. The method according to any one of claims 9 to 13, wherein the temperature for the culture in the step (b) is at 38°

C.

15. The method according to any one of claims 9 to 14, wherein the conditions for the culture in the step (b) are under conditions at 5% CO₂, 5% O₂ and 90% N₂.

16. The method according to any one of claims 9 to 15, wherein the in vitro culture in the step (b) is performed using CMRL-1066.

17. An established cynomolgus monkey-derived cell having the following properties:

(i) being capable of indefinitely proliferating while maintaining an undifferentiated state;

(ii) having the same chromosome number as that of original Macaca fascicularis;

(iii) exhibiting the pluripotency when transplanted to hypodermis, the renal capsule or the testis of 8-12 week-old SCID mouse or nude mouse;

(iv) being negative for SSEA-1 and positive for SSEA-3 and SSEA-4; and

(v) exhibiting an alkaline phosphatase activity.

18. The established cynomologus monkey-derived cell according to claim 17, wherein the cell exhibits the ability to differentiate into at least one selected from the group consisting of ectoderm, mesoderm and endoderm, when transplanted to hypodermis, the renal capsule or the testis of 8-12 week-old SCID mouse or nude mouse.

19. The established cynomologus monkey-derived cell according to claim 17 or 18, wherein the cell exhibits the ability to differentiate into at least one selected from the group consisting of neuron, glia, muscle, cartilage, bone, ciliated epithelium and intestinal epithelium, when transplanted to hypodermis, the renal capsule or the testis of a 8-12 week-old SCID mouse or nude mouse.