Method of making a population of chondrocytes from reprogrammed chondrocytes

• US 10,385,318 B2
• Filed: 01/16/2018
• Issued: 08/20/2019
• Est. Priority Date: 10/29/2012
• Status: Active Grant

• Current Assignee: Scripps Health
• Original Assignee: Scripps Health
• Inventors: D'Lima, Darryl D., Olee, Tsaiwei, Colwell, Clifford W.
• Primary Examiner(s): Wilson, Michael C
• Application Number:
  US15/872,577
• Publication Number:
  US 20180155694A1
• Time in Patent Office: 581 Days
• Field of Search: 435325, 435455
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• 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: A61P 35/00 Antineoplastic agents
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2506/13 from connective tissue cells, from mesenchymal cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Disclosed herein are methods of producing induced pluripotent stem cells from chondrocytes, and further, methods of producing chondrocytes from said induced pluripotent stem cells. The invention further provides methods of regenerating cartilaginous tissue.

Claims

1. A method of generating a population of chondrocytes, comprising:

a) introducing at least one nucleic acid encoding an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, into isolated chondrocytes;

b) culturing the isolated chondrocytes of step a) in the presence of pigment epithelium-derived factor (PEDF) such that colonies of cells expressing Oct4, Rex1, SSEA4, Tra1-60, and Tra1-81 are obtained;

c) producing cell aggregates from the colonies;

d) differentiating the cell aggregates such that chondroprogenitors are obtained;

e) differentiating the chondroprogenitors such that a population of chondrocytes is obtained.

2. The method of claim 1, further comprising culturing the isolated chondrocytes of step a) in the absence of basic fibroblast growth factor (bFGF).

3. The method of claim 1, wherein producing cell aggregates comprises mechanical dissection of the colonies.

4. The method of claim 1, wherein producing cell aggregates does not comprise enzymatic digestion of the colonies.

5. The method of claim 1, wherein differentiating the cell aggregates comprises culturing the cell aggregates in a tissue culture-treated vessel.

6. The method of claim 5, wherein the tissue culture-treated vessel is plastic.

7. The method of claim 1, wherein the chondroprogeniters are embedded in a matrix gel material comprising at least one of a proteoglycan, glycosoaminoglycan, fiber, and collagen.

8. The method of claim 1, wherein the chondroprogenitors are differentiated in the presence of TGF-β

3.

9. The method of claim 1, wherein the chondroprogenitors are differentiated in the absence of TGF-β

3.

10. The method of claim 1, wherein the chondroprogenitors are differentiated in the three-dimensional culture in the presence of at least one of BMP2 or BMP4.

11. The method of claim 1, wherein the chondroprogenitors are differentiated in the presence of at least one of a collagen, proteoglycan, fibrin, hyaluronic acid, poly-D-lactide, poly-L-lactide, poly-DL-lactide, polyglycolic acid, polylactic acid, hydroxyapatite, calcium phosphate, atelocollagen, fibrin, alginate, agar, gelatin, glycosaminoglycan (GAG), TGF-β

3, TGF-β

1, IGF-1, or bFGF.

12. The method of claim 11, wherein the chondroprogenitors are differentiated in the presence of at least one of collagen, proteoglycan, fibrin, or hyaluronic acid.

13. The method of claim 11, wherein the chondroprogenitors are differentiated in the presence of collagen selected from the group consisting of type H collagen and type VI collagen.

14. The method of claim 13, wherein the collagen is cross-linked.

15. The method of claim 13, wherein the collagen is solubilized.

16. The method of claim 11, wherein the chondroprogenitors are differentiated in the presence of proteoglycan.

17. The method of claim 1, wherein the method does not comprise formation of an embryoid body.

18. The method of claim 1, wherein producing the cell aggregates in step c) comprises formation of embryoid bodies.