Compositions of mesenchymal stem cells to regenerate bone

• US 9,511,093 B2
• Filed: 03/23/2010
• Issued: 12/06/2016
• Est. Priority Date: 03/23/2009
• Status: Active Grant

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Gregory, Carl A., Prockop, Darwin J.
• Primary Examiner(s): Visone, Thomas J
• Application Number:
  US12/730,022
• Publication Number:
  US 20100247494A1
• Time in Patent Office: 2,450 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)

Abstract

The present invention encompasses an osteogenic composition comprising mesenchymal stem cells pre-cultured in the presence of an agent that accelerates canonical Wnt signaling therein. Also, provided are osteogenic compositions incorporated into a biocompatible gel. The present invention provides methods for treating bone degeneration or injury associated with a pathophysiological condition in a mammal or for accelerating repair of a skeletal injury in a mammal by administering to the mammal or contacting the site of injury with the osteogenic composition.

Claims

1. A population of pre-conditioned mesenchymal stem cells, wherein the population of pre-conditioned mesenchymal stem cells is produced by:

culturing mesenchymal stem cells in a conditioning medium lacking dexamethasone and comprising an effective amount of an inhibitor of peroxisome proliferator-activated receptor γ

(PPAR-γ

) for 5 to 14 days so as;

(a) to inhibit regulation of adipogenesis in the mesenchymal stem cells,

(b) to induce the mesenchymal stem cells to exhibit;

(i) early or primitive osteoblastic characteristics without undergoing terminal differentiation,

(ii) an upregulation in gene expression for collagen types I, III, V, VI, XI, and XII, and

(iii) downregulation or depletion of cytosolic glycogen synthase kinase 3β

, and

(c) to not induce mineralization,

wherein the pre-conditioned mesenchymal stem cells exhibit (a), (b), and (c) when removed from the conditioning medium.

2. The population of pre-conditioned mesenchymal stem cells of claim 1, further comprising one of either a biocompatible lattice and a biocompatible gel.

3. The population of pre-conditioned mesenchymal stem cells of claim 2, wherein the biocompatible lattice is clotted plasma.

4. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein said inhibitor is 2-chloro-5-nitro-N-phenyl-benzamide (GW9662) provided in an amount of about 10 μ

M.

5. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein said mesenchymal stem cells are retrieved from at least one of the group consisting of adult bone marrow, adipose tissue, peripheral blood, umbilical cord blood, synovial membrane, and periodontal ligament.

6. The population of pre-conditioned mesenchymal stem cells of claim 5, wherein said inhibitor of peroxisome proliferator-activated receptor γ

is 2-chloro-5-nitro-N-phenyl-benzamide (GW9662).

7. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein said conditioning medium further comprises beta glycerophosphate and ascorbate.

8. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein said conditioning medium is provided after the mesenchymal stem cells have been allowed to divide in vitro for about six to eight doublings.

13. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein the conditioning medium is provided for about eight to ten days with periodic changes in the conditioning medium.

15. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein the mesenchymal stem cells are initially seeded in vitro at a density of up to about 100 cells/cm² and allowed to divide for six to eight doublings without the conditioning medium.

17. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein the conditioning medium further comprises about 5 mM beta-glycerophosphate and about 50 μ

g/mL ascorbate-2-phosphate.

18. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein the pre-conditioned mesenchymal stem cells exhibit reduced secretion in dickkopf-1.

19. The population of pre-conditioned mesenchymal stem cells of claim 1, wherein the pre-conditioned mesenchymal stem cells further exhibit reduced secretion of one or more inflammatory mediators, including at least one of IL-1, IL-8, and chemokines of a CXCL family, the reduced secretion being compared to non pre-conditioned mesenchymal stem cells.

9. A composition comprising:

a biocompatible lattice; and

a population of pre-conditioned mesenchymal stem cells comingled with the biocompatible lattice, wherein the population of pre-conditioned mesenchymal stem cells is produced by;

culturing mesenchymal stem cells in a conditioning medium lacking dexamethasone and comprising an effective amount of an inhibitor of peroxisome proliferator-activated receptor γ

(PPAR-γ

) for 5 to 14 days so as;

(a) to inhibit regulation of adipogenesis in the mesenchymal stem cells,

(b) to induce the mesenchymal stem cells to exhibit;

(i) early or primitive osteoblastic characteristics without undergoing terminal differentiation,

(ii) an upregulation in gene expression for collagen types I, III, V, VI, XI, and XII, and

(iii) downregulation or depletion of cytosolic glycogen synthase kinase 3β

, and

(c) to not induce mineralization,

wherein the pre-conditioned mesenchymal stem cells exhibit (a), (b), and (c) when removed from the conditioning medium.

10. The composition of claim 9, wherein said pre-conditioned mesenchymal stem cells are suspended in a biologically compatible diluent prior to comingling with the biocompatible lattice.

11. The composition of claim 9, wherein said composition is stored.

12. A composition comprising:

a three-dimensional scaffold; and

a population of pre-conditioned mesenchymal stem cells comingled with the three-dimensional scaffold, wherein the population of pre-conditioned mesenchymal stem cells is produced by;

culturing mesenchymal stem cells in a conditioning medium lacking dexamethasone and comprising an effective amount of an inhibitor of peroxisome proliferator-activated receptor γ

(PPAR-γ

) for 5 to 14 days so as;

(a) to inhibit regulation of adipogenesis in the mesenchymal stem cells,

(b) to induce the mesenchymal stem cells to exhibit;

(i) early or primitive osteoblastic characteristics without undergoing terminal differentiation,

(ii) an upregulation in gene expression for collagen types I, III, V, VI, XI, and XII, and

(iii) downregulation or depletion of cytosolic glycogen synthase kinase 3β

, and

(c) to not induce mineralization,

wherein the pre-conditioned mesenchymal stem cells exhibit (a), (b), and (c) when removed from the conditioning medium.

14. The composition of claim 12, wherein the pre-conditioned mesenchymal stem cells are initially self-renewed and expanded by dividing without the conditioning medium for six to eight doublings in vitro.

16. The composition of claim 12, wherein the pre-conditioned mesenchymal stem cells seeded on the three-dimensional scaffold are suspended in a biologically compatible diluent prior to utilization.