Stem cell populations and methods of use

• US 20050272152A1
• Filed: 05/13/2005
• Published: 12/08/2005
• Est. Priority Date: 05/14/2004
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Deluca, Keith, Liebmann-Vinson, Andrea, Xu, Ruiling
• Granted Patent: US 7,790,458 B2
• Time in Patent Office: Days
• US Class Current: 435/372
• 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

The invention provides populations of expanded CD34-expressing cells and methods of use. Particular embodiments provide for defined culture media useful for growing these cells, and grafts comprising these cells. The invention finds use in methods for reconstituting, repairing, and regenerating tissue damage.

Claims

1. A population of stem and progenitor cells that has been expanded in a cytokine cocktail, wherein said population comprises a CD34⁺ cell population and a CD34^br cell population, and wherein said CD34^br cell population has an antibody bound per cell value that is at least 1.5-fold greater than the antibody bound per cell value for said population of CD34⁺ cells.

2. The population of claim 1, wherein said cocktail comprises at least 8 cytokines selected from the group consisting of fibronectin, SDF-1α

, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, bFGF, angiotensin, PDGF-AB, TGF-β

1, GM-CSF, VEGF, EGF, M-CSF, and MCP-1, PlGF, hematopoietic adhesion peptide comprising the amino acid sequence VTCG, TRANCE, G-CSF, Tpo, LIF, and SCGF-α

, and combinations thereof.

3. The population of claim 2, wherein said cocktail is G2, F2, D6, F2N, F4, or G8.

4. The population of claim 3, wherein a substantial proportion of the cells are hematopoietic progenitor cells.

5. The population of claim 3, wherein a substantial proportion of the cells are endothelial progenitor cells.

6. The population of claim 3, wherein a substantial proportion of the cells are hemangioblasts.

7. The population of claim 3, wherein a substantial proportion of the cells comprise stem and progenitor cells selected from the group consisting of BFU-E/CFU-E, CFU-GM, CFU-G, CFU-GEMM, and combinations thereof.

8. The population of claim 3, wherein a substantial proportion of the cells express CD31, CD33, or CD71, or combinations thereof.

9. The population of claim 3, wherein greater than 5% of the cells are CD34^br cells.

10. The population of claim 3, wherein a percentage of expanded cells express cell surface markers selected from the group consisting of less than 10% expressing CD14, less than 1% expressing CD16, less than 1% expressing CD3, greater than 50% expressing CD31, greater than 50% expressing CD33, greater than 10% expressing CD34, less than 10% expressing CD38, greater than 40% expressing CD71, greater than 5% expressing CD133, and combinations thereof.

11. A cell culture medium for expanding a population of stem and progenitor cells, said medium comprising at least 8 cytokines selected from the group consisting of fibronectin, SDF-1α

, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, bFGF, angiotensin, PDGF-AB, TGF-β

1, GM-CSF, VEGF, EGF, M-CSF, and MCP-1, PlGF, hematopoietic adhesion peptide comprising the amino acid sequence VTCG, TRANCE, G-CSF, Tpo, LIF, and SCGF-α

, and combinations thereof.

12. The cell culture medium of claim 11, wherein said cytokines comprise fibronectin, SDF-1α

, 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.

13. A method of producing an expanded population of stromal cells, said method comprising culturing cord blood-derived total nucleated cells (TNCs) in the cell culture medium of claim 12.

14. The cell culture medium of claim 11, wherein said cytokines comprise angiotensin, IL-6, SCF, BDNF, IL-3, PDGF-AB, BMP-4, TGF-β

1, thrombospondin, GM-CSF, VEGF, EGF, bFGF, M-CSF, and MCP-1.

15. The cell culture medium of claim 11, wherein said cytokines comprise PlGF, SDF-1α

, IL-6, SCF, IL-5, BDNF, IL-11, IL-3, EPO, PDGF-AB, Flt-3/Flk-2 ligand, hematopoietic adhesion peptide comprising the amino acid sequence VTCG, TRANCE, GM-CSF, VEGF, and G-CSF.

16. A method of expanding and differentiating a population of stem and progenitor cells, said method comprising culturing said population in the cell culture medium of claim 15.

17. The method of claim 16, wherein said culture medium provides for differentiation of at least a portion of said population into erythrocytes.

18. The cell culture medium of claim 11, wherein said cytokines comprise BDNF, bFGF, EPO, fibronectin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-3, IL-5, SCF, TGF-β

1, G-CSF, and GM-CSF.

19. The cell culture medium of claim 11, wherein said cytokines comprise bFGF, EPO, Flt-3/Flk-2 ligand, IL-11, IL-6, PD-ECGF, thrombospondin, and Tpo.

20. The cell culture medium of claim 11, wherein said cytokines comprise BDNF, bFGF, Flt-3/Flk-2 ligand, IL-3, IL-6, PD-ECGF, SCF, G-CSF, LIF, and SCGF-α

.

21. A method of producing an expanded population of stem and progenitor cells, said method comprising culturing a population of stem and progenitor cells in a cell culture medium comprising at least 8 cytokines selected from the group consisting of fibronectin, SDF-1α

, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, bFGF, angiotensin, PDGF-AB, TGF-β

1, GM-CSF, VEGF, EGF, M-CSF, and MCP-1, PlGF, hematopoietic adhesion peptide comprising the amino acid sequence VTCG, TRANCE, G-CSF, Tpo, LIF, and SCGF-α

, and combinations thereof.

22. The method of claim 21, wherein said population comprises a CD34⁺ cell population and a CD34^br cell population, and wherein said CD34^br cell population has an antibody bound per cell value that is at least 1.5-fold greater than the antibody bound per cell value for said population of CD34⁺ cells

23. A method of transplanting a population of stem and progenitor cells comprising administering an expanded population of stem and progenitor cells to a patient in need thereof, wherein said expanded population comprises a CD34⁺ cell population and a CD34^br cell population, and wherein said CD34^br cell population has an antibody bound per cell value that is at least 1.5-fold greater than the antibody bound per cell value for said population of CD34⁺ cells.

24. The method of claim 23, wherein said expanded population of stem and progenitor cells is administered in an amount effective to restore the production of lymphoid or myeloid cells.

25. The method of claim 23, wherein said expanded population of stem and progenitor cells is administered in an amount effective to promote vascularization at a site in said patient.

26. The method of claim 25, wherein said site is a damaged tissue.

27. The method of claim 26, wherein said tissue is heart tissue.

28. The method of claim 23, wherein said expanded population of stem and progenitor cells is impregnated into a biocompatible scaffold prior to transplantation.

29. The method of claim 23, wherein said expanded population of stem and progenitor cells is administered in an amount effective to repair a damaged tissue.

30. The method of claim 29 wherein said tissue is heart tissue.

31. The method of claim 29, wherein said expanded population of stem and progenitor cells is impregnated into a biocompatible scaffold prior to transplantation.

32. The method of claim 23, wherein said population of stem and progenitor cells are transplanted in an amount effective to provide engraftment of megakaryocytes to a patient in need thereof.