Cell culture environments for the serum-free expansion of mesenchymal stem cells

• US 20050265980A1
• Filed: 05/13/2005
• Published: 12/01/2005
• Est. Priority Date: 05/14/2004
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Chen, Chang, Mitchell, Matthew, Rowley, Jonathan Allen, Liebmann-Vinson, Andrea, Haaland, Perry D., Xu, Ruiling, Chaney, Bryce N.
• Application Number:
  US11/129,612
• Publication Number:
  US 20050265980A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/42 Organic phosphate, e.g. beta glycerophosphate
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/21 Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 : Interleukins [IL]
• CPC Class Codes: C12N 2501/2303 : Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2305 : Interleukin-5 (IL-5)
• CPC Class Codes: C12N 2501/2306 : Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2311 : Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/235 : Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/26 : Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/32 : Angiotensins [AT], angiotensinogen
• CPC Class Codes: C12N 2501/39 : Steroid hormones
• CPC Class Codes: C12N 2501/415 : Wnt; Frizzeled
• CPC Class Codes: C12N 2501/58 : Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/60 : Transcription factors
• CPC Class Codes: C12N 2501/70 : Enzymes
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2533/52 : Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 : Collagen; Gelatin
• CPC Class Codes: C12N 2533/70 : Polysaccharides
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0663 : Bone marrow mesenchymal stem cells (BM-MSC)

Abstract

Compositions and methods for promoting mesenchymal stem cell expansion while maintaining a pluripotent phenotype are disclosed. Serum-free cell culture systems and kits and methods of use for mesenchymal stem cell expansion are provided. Methods also comprise the use of the expanded mesenchymal stem cells to treat various disorders or diseases, particularly those of the cardiovascular system, bone, or cartilage.

Claims

1. A serum-free cell culture system comprising a serum-free cell culture medium and a cell culture surface that promotes the adhesion and expansion of mesenchymal stem cells, wherein at least one insoluble substrate protein is presented from said cell culture surface, and wherein said serum-free cell culture medium is selected from the group consisting of:

a) a medium comprising bFGF in combination with at least one growth factor selected from the group consisting of a WNT signaling agonist, TGF-β

, and EGF;

b) a medium comprising a WNT signaling agonist in combination with at least one growth factor selected from the group consisting of bFGF, TGF-β

, and EGF;

c) a medium comprising TGF-β

in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and EGF;

d) a medium comprising EGF in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and TGF-β

;

e) a medium comprising fibronectin, SDF-1 α

, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF;

f) a medium comprising BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α

, and a WNT signaling agonist;

g) a medium comprising bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist;

h) a medium comprising bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist;

i) a medium comprising bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α

, and a WNT signaling agonist;

j) a medium comprising bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and a WNT signaling agonist;

k) a medium comprising bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT signaling agonist; and

l) a medium comprising bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and a WNT signaling agonist.

2. The serum-free cell culture system of claim 1, wherein said WNT signaling agonist is BIO or WNT-3a.

3. The serum-free cell culture system of claim 1, wherein the insoluble substrate protein is an extracellular matrix protein.

4. The serum-free cell culture system of claim 3, wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.

5. The serum-free cell culture system of claim 4, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.

6. The serum-free cell culture system of claim 5, wherein the WNT signaling agonist is BIO or WNT-3a, and wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material.

7. A method of expanding a population of mesenchymal stem cells using the cell culture system of claim 1.

8. The method of claim 7, wherein said WNT signaling agonist is BIO or WNT-3a, wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material, and wherein the insoluble substrate protein is a combination of collagen I and fibronectin.

9. An expanded population of mesenchymal stem cells that has never been exposed to serum, wherein said population is produced by the method of claim 7.

10. A method of transplanting an expanded population of mesenchymal stem cells, comprising administering the population of claim 9 to a patient in need thereof, wherein the population is administered in an amount effective to reconstitute cardiovascular, bone, or cartilage tissue.

11. A kit comprising the serum-free cell culture system of claim 1.

12. The kit of claim 11, wherein said WNT signaling agonist is BIO or WNT-3a, wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material, and wherein the insoluble substrate protein is a combination of collagen I and fibronectin.

13. A serum-free cell culture system comprising a serum-free cell culture medium and a cell culture surface, wherein said cell culture surface promotes the adhesion and expansion of mesenchymal stem cells and comprises a cell culture support bound to a cell adhesion resistant material, and wherein at least one insoluble substrate protein is presented from the cell culture surface.

14. The serum-free cell culture system of claim 13, wherein the insoluble substrate protein is an extracellular matrix protein.

15. The serum-free cell culture system of claim 14, wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.

16. The serum-free cell culture system of claim 15, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.

17. The serum-free cell culture system of claim 13, wherein said serum-free cell culture medium is selected from the group consisting of:

a) a medium comprising bFGF in combination with at least one growth factor selected from the group consisting of a WNT signaling agonist, TGF-β

, and EGF;

b) a medium comprising a WNT signaling agonist in combination with at least one growth factor selected from the group consisting of bFGF, TGF-β

, and EGF;

c) a medium comprising TGF-β

in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and EGF;

d) a medium comprising EGF in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and TGF-β

;

e) a medium comprising fibronectin, SDF-1 α

, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF;

f) a medium comprising BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α

, and a WNT signaling agonist;

g) a medium comprising bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist;

h) a medium comprising bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist;

i) a medium comprising bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α

, and a WNT signaling agonist;

j) a medium comprising bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and a WNT signaling agonist;

k) a medium comprising bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT signaling agonist; and

l) a medium comprising bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and a WNT signaling agonist.

18. The serum-free cell culture system of claim 17, wherein said WNT signaling agonist is BIO or WNT-3a.

19. The serum-free cell culture system of claim 17, wherein the insoluble substrate protein is an extracellular matrix protein and wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.

20. The serum-free cell culture system of claim 19, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.

21. A method of expanding a population of mesenchymal stem cells using the cell culture system of claim 13.

22. The method of claim 21, wherein the insoluble substrate protein is a combination of collagen I and fibronectin.

23. An expanded population of mesenchymal stem cells, said population produced by the method of claim 21, wherein said cells were unexposed to serum.

24. A method of transplanting an expanded population of mesenchymal stem cells, comprising administering the population of claim 23 to a patient in need thereof, wherein the population is administered in an amount effective to reconstitute cardiovascular, bone, or cartilage tissue.

25. A kit comprising the serum-free cell culture system of claim 13.

26. The kit of claim 25, wherein the insoluble substrate protein is a combination of collagen I and fibronectin.