Use of adipose tissue-derived stromal cells for chondrocyte differentiation and cartilage repair

• US 6,429,013 B1
• Filed: 05/17/2000
• Issued: 08/06/2002
• Est. Priority Date: 08/19/1999
• Status: Expired due to Term

• Current Assignee: Cognate BioServices, Inc.
• Original Assignee: Artecel Science Incorporated
• Inventors: Halvorsen, Yuan-Di C., Gimble, Jeffrey Martin, Wilkison, William O.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Davis, Katharine F
• Application Number:
  US09/573,989
• Time in Patent Office: 811 Days
• Field of Search: 435/325, 435/366, 435/377, 435/455, 435/395, 424/93.21
• US Class Current: 435/377
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61L 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: C12N 2500/30 Organic components
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/1305 Adipocytes
• CPC Class Codes: C12N 2506/1384 from adipose-derived stem cells [ADSC], from adipose stromal stem cells
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage are disclosed. The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.

Claims

1. A composition comprising:

(a) an isolated human adipose tissue derived stromal cell; and

(b) a chemically defined culture medium having (i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype (ii) an antibiotic (iii) a nutrient supplement (iv) ascorbate or related vitamin C analogue and (v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor.

2. The composition of claim 1, wherein the chondroinductive agent is selected individually or in combination from the group consisting of:

a glucocorticoid;

a member of the transforming growth factor-beta superfamily;

a collagenous extracellular matrix molecule; and

a vitamin A analog.

3. The composition of claim 1, wherein said antibiotic is penicillin.

4. The composition of claim 1, wherein said antibiotic is streptomycin.

5. The composition of claim 2, wherein the chondroinductive agent is hydrocortisone.

6. The composition of claim 2, wherein the chondroinductive agent is dexamethasone.

7. The composition of claim 2, wherein the concentration of dexamethasone is about 1 to about 100 nM.

8. The composition of claim 2, wherein said transforming growth factor-beta is selected from the group consisting of:

bone morphogenic protein-2, bone morphogenic protein-4, TGF-β

1 (transforming growth factor-beta-1), TGF-β

2 (transforming growth factor-beta-2), TGF-β

3 (transforming growth factor-beta-3), IGF (insulin-like growth factor), PDGF (platelet-derived growth factor), EGF (epidermal growth factor), aFBF (acidic fibroblast growth factor), bFBF (basic fibroblast growth factor), HGF (hepatocytic growth factor), KGF (keratocyte growth factor), inhibin A, and chondrogenic stimulating factor.

9. The composition of claim 2, wherein the collagenous extracellular matrix molecule is collagen I.

10. The composition of claim 2, wherein the vitamin A analog is retinoic acid.

11.The composition of claim 3, wherein the concentration of penicillin is about 10 to about 200 units per ml.

11. The composition of claim 4, wherein the concentration of streptomycin is about 10 to about 200 μ

g per ml.

12. The composition of claim 5, wherein the concentration of hydrocortisone is about 1 to about 100 nM.

13. The composition of claim 8, wherein the concentration of transforming growth factor-beta is about 1 to about 100 ng per ml.

14. The composition according to claim 8, wherein the concentration of TGF-β

1 is about 1 ng/ml to about 10 ng/ml.

15. The composition of claim 10, wherein the concentration of retinoic acid is about 0.1 ng per ml to about 1 μ

g per ml.

16. A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:

a) pelleting said stromal cells and preadipocytes by centrifuging between 50,000 to 5 million cells at 500×

g for 2 to 20 minutes in sterile tubes containing a medium;

b) plating isolated stromal cells and preadipocytes at a density of 500 to 20,000 cells/cm² in a differentiating medium;

c) supplementing said medium with;

i) a chondroinductive agent capable of activating any cellular signal transduction pathway leading to the mature chondrocyte phenotype

ii) an antibiotic

iii) a nutrient supplement with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins

iv) ascorbate or related vitamin C analog

v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and

d) incubating said cells at about 31°

C. to 37°

C. for about 3-4 weeks with 5% CO₂ and between 1% and 20% oxygen; and

,

e) determining the extent of differentiation of the isolated cells.

17. A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:

a) suspending stromal cells and preadipocytes at a concentration of 0.5 to 10 million cells per ml in calcium alginate or any other biocompatible lattice or matrix capable of supporting chondrogenesis in a three-dimensional configuration;

b) transferring cells to 35 mm culture dishes and plating cells at a density of 500 to 20,000 cells/cm² in a differentiating medium comprising a chemically defined culture medium having or supplemented with;

i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype;

ii) an antibiotic;

iii) a nutrient supplement with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;

iv) ascorbate or related vitamin C analog;

v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and

c) incubating said cells at about 31 to 37°

C. for about 3-4 weeks in an incubator with 5% CO₂ and between 1% and 20% oxygen, and

d) determining the extent of differentiation of the isolated cells.

18. A composition comprising:

a) an isolated adipose tissue derived stromal cell that has been induced to express at least one characteristic of a chondrocyte; and

b) a biocompatible matrix.

19. The composition of claim 19, wherein the matrix is capable of supporting chondrogenesis.

20. The composition of claim 19, wherein the matrix is selected from the group consisting of calcium alginate, agarose, fibrin, and collagen.

21. An isolated adipose tissue derived stromal cell that has been induced to express at least one characteristic of a chondrocyte wherein a foreign nucleic acid has been introduced into the cell.

22. A synthetic cartilage patch comprising an isolated adipose tissue derived stromal cell that has been induced to express at least one characteristic of a chondrocyte.

23. The cartilage patch of claim 23, wherein the patch is grown in pre-shaped culture wells to produce a patch of specific dimensions.

24. The cartilage patch of claim 23, wherein the patch is grown in culture wells coated with a molecule that discourages adhesion of chondrogenic cells to the cell contacting surface.

25. The culture wells of claim 24, wherein the culture wells have a cross-sectional surface area of about 25 cm².

26. The culture wells of claim 24, wherein the culture wells have a depth large enough to form a cartilage patch of about 0.3 cm.

27. The cartilage patch of claim 25, wherein the coating molecule is selected from the group consisting of dichlorodimethylsilane and polytetrafluoroethylene based reagents.

28. The cartilage patch of claim 23, wherein the culture wells are cast from a pliable or moldable biocompatible material that does not permit the attachment of cells.

29. The culture wells of claim 29, wherein the pliable or moldable biocompatible material is selected from the group consisting of agarose, glass, untreated cell culture plastic, and polytetrafluoroethylene.

30. An isolated human adipose tissue derived stromal cell that has been induced to express at least one characteristic of a chondrocyte.

31. The cell of claim 31, implanted in vivo.