In vitro chondrogenic induction of human mesenchymal stem cells

• US 5,908,784 A
• Filed: 11/15/1996
• Issued: 06/01/1999
• Est. Priority Date: 11/16/1995
• Status: Expired due to Term

• Current Assignee: Case Western Reserve University, James E. Bailey, Pauli T. Kallio, Peter Brunker, Wofgang Minas
• Original Assignee: Case Western Reserve University
• Inventors: Yoo, Jung, Johnstone, Brian
• Primary Examiner(s): Saunders, David
• Assistant Examiner(s): VANDERVEGT, FRANCOIS P
• Application Number:
  US08/749,484
• Time in Patent Office: 928 Days
• Field of Search: 435/395, 435/397, 435/405, 435/372, 435/377, 530/356
• US Class Current: 435/372
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Disclosed are a composition of chemically defined components which support the in vitro chondrogenesis of mesenchymal progenitor cells, a method for in vitro chondrogenic induction of such progenitor cells and a method of forming human chondrocytes in vitro from such progenitor cells.

Claims

1. A composition for the in vitro chondrogenesis of human mesenchymal precursor cells and the in vitro formation of human chondrocytes therefrom, which composition comprises isolated human mesenchymal stem cells condensed into close proximity as a packed cell pellet and at least one chondroinductive agent in contact therewith.

2. The composition of claim 1 wherein the mesenchymal stem cells are isolated, culture expanded human mesenchymal stem cells.

3. The composition of claim 1 wherein the mesenchymal stem cells are in a chemically defined serum-free environment.

4. The composition of claim 1 wherein the chondroinductive agent is selected from the group consisting of (i) a glucocorticoid;

(ii) a member of the transforming growth factor-β

superfamily; and

(iii) a component of the collagenous extracellular matrix.

5. The composition of claim 4 wherein the glucocorticoid is dexamethasone.

6. The composition of claim 4 wherein the member of the transforming growth factor superfamily is selected from the group consisting of a bone morphogenic protein, TGF-β

1, inhibin A and chondrogenic stimulating activity factor.

7. The composition of claim 6 wherein the bone morphogenic protein is BMP-4.

8. The composition of claim 4 wherein the component of the collagenous extracellular matrix is collagen I.

9. The composition of claim 8 wherein the collagen I is in the form of a gel.

10. The composition of claim 1 wherein the chondroinductive agent is a combination of dexamethasone and TGF-β

1.

11. A process for producing chondrocytes from mesenchyrnal stem cells by contacting mesenchymal stem cells with a chondroinductive agent in vitro wherein the stern cells are condensed into close proximity as a packed cell pellet.

12. The process of claim 11 wherein the mesenchymal stem cells are isolated, culture expanded human mesenchymal stem cells.

13. The process of claim 11 wherein the mesenchymal stem cells are in a chemically defined serum-free environment.

14. The process of claim 11 wherein the chondroinductive agent is selected from the group consisting of (i) a glucocorticoid;

(ii) a member of the transforming growth factor-β

superfamily; and

(iii) a component of the collagenous extracellular matrix.

15. The process of claim 11 wherein the chondroinductive agent is a combination of dexamethasone and TGF-β

1.

16. The process of claim 11 wherein the step of contacting comprises culturing a pellet of human mesenchymal stem cells in a chemically defined serum-free medium.

17. The process of claim 16 wherein the chemically defined serum-free medium comprises (1) a chemically defined minimum essential medium;

(2) ascorbate or an analog thereof;

(3) an iron source;

(4) insulin or an insulin-like growth factor; and

(5) at least one chondroinductive agent or factor.

18. The method of claim 11 wherein the cells are cultured with the chondroinductive composition and thereafter placed in a rigid porous vessel.

19. The method of claim 18 wherein the rigid porous vessel is a ceramic cube.

20. A process for inducing chondrogenesis in mesenchymal stem cells by contacting mesenchymal stem cells with a chondroinductive agent in vitro wherein the stem cells are condensed into close proximity as a packed cell pellet.

21. The process of claim 20 wherein the mesenchymal stem cells are isolated, culture expanded human mesenchymal stem cells.

22. The process of claim 20 wherein the mesenchymal stem cells are in a chemically defined serum-free environment.

23. The process of claim 20 wherein the chondroinductive agent is selected from the group consisting of (i) a glucocorticoid;

(ii) a member of the transforming growth factor-β

superfamily; and

(iii) a component of the collagenous extracellular matrix.

24. The process of claim 20 wherein the chondroinductive agent is a combination of dexamethasone and TGF-β

1.

25. The process of claim 20 wherein the step of contacting comprises culturing a pellet of human mesenchymal stem cells in a chemically defined serum-free medium.

26. The process of claim 25 wherein the chemically defined serum-free medium comprises (1) a chemically defined minimum essential medium;

(2) ascorbate or an analog thereof;

(3) an iron source;

(4) insulin or an insulin-like growth factor; and

(5) at least one chondroinductive agent or factor.

27. The method of claim 20 wherein the cells are cultured with the chondroinductive composition and thereafter placed in a rigid porous vessel.

28. The method of claim 27 wherein the rigid porous vessel is a ceramic cube.