Umbilical cord lining stem cells and methods and material for isolating and culturing same

• US 8,778,679 B2
• Filed: 11/02/2012
• Issued: 07/15/2014
• Est. Priority Date: 09/23/2009
• Status: Active Grant

• Current Assignee: Restem LLC
• Original Assignee: DaVinci Biosciences LLC
• Inventors: Silva, Francisco J., Gonzalez, Rafael
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): CHONG, STEPHEN M
• Application Number:
  US13/668,138
• Publication Number:
  US 20130065302A1
• Time in Patent Office: 620 Days
• Field of Search: None
• US Class Current: 435/374
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

Human umbilical cord lining stem cells that are capable of differentiating into cells of the mesodermal lineage and ectodermal lineage are described, as well as methods of isolating, expanding, culturing, and cryopreserving such cells.

Claims

1. A method for isolating umbilical cord lining stem cells (ULSCs) from an umbilical cord, said method comprising

a) obtaining the lining of an umbilical cord, wherein said lining is substantially free of blood, venous tissue, and arterial tissue;

and

b) culturing explants of said lining on a fibronectin-coated solid substrate in the presence of a low glucose growth medium for a period of time sufficient for said ULSCs to adhere to said fibronectin-coated solid substrate, said growth medium comprising 15% fetal bovine serum, a stabilized dipeptide of L-alanyl-L-glutamine, antibiotic, and a growth factor selected from the group consisting of basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF).

2. The method of claim 1, said growth medium further comprising insulin, transferrin, selenium, and sodium pyruvate.

3. The method of claim 2, said growth medium further comprising putrescine.

4. The method of claim 3, said growth medium comprising bFGF, LIF, and EGF.

5. The method of claim 1, wherein said antibiotic is gentamycin.

6. The method of claim 1, wherein said antibiotic is penicillin and streptomycin.

7. The method of claim 1, wherein the upper surface of each said explant is in contact with a solid substrate.

8. The method of claim 1, said method further comprising washing said cells adhered to said fibronectin-coated solid substrate.

9. A composition for culturing umbilical cord lining stem cells (ULSCs), said composition comprising:

a) a low glucose growth medium;

b) 10% to 20% serum;

c) 0.7 to 1.5% of a stabilized dipeptide of L-alanyl-L-glutamine;

d) 1 to 100 ng/mL of a growth factor selected from the group consisting of basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF); and

e) 1 to 3% of an antibiotic.

10. The composition of claim 9, further comprising:

f) 0.1 mg/mL to 100 mg/mL of insulin;

g) 0.1 mg/mL to 100 mg/mL of transferrin;

h) 0.1 μ

g/mL to 100 μ

g/mL of selenium; and

i) 0.5 to 1.5% sodium pyruvate.

11. The composition of claim 10, further comprising 0.05 μ

g/mL to 100 μ

g/mL of putrescine.

12. The composition of claim 10, said composition comprising 15% serum;

1% of said stabilized dipeptide of L-alanyl-L-glutamine;

10 ng/mL of bFGF, 10 ng/mL of LIF;

1% of said antibiotic;

10 ng/mL of insulin;

0.55 mg/mL of transferrin; and

0.5 μ

g/mL of selenium.

13. The composition of claim 12, further comprising 10 μ

g/mL of putrescine and 10 ng/mL of EGF.

14. A composition comprising a purified population of ULSCs and a culture medium, wherein said culture medium comprises a low glucose growth medium;

10% to 20% serum;

0.7 to 1.5% of a stabilized dipeptide of L-alanyl-L-glutamine;

1 to 100 ng/mL of a growth factor selected from the group consisting of bFGF, LIF, and EGF; and

1 to 3% of an antibiotic, wherein said ULSCs are positive for CD105, CD106, CD90, CD73, SSEA-4, and STRO-1, and negative for CD45, CD34, CD19, and HLA-DR.

15. The composition of claim 14, wherein said culture medium further comprises 0.1 mg/mL to 100 mg/mL of insulin;

0.1 mg/mL to 100 mg/mL of transferrin;

0.1 μ

g/mL to 100 μ

g/mL of selenium; and

0.5 to 1.5% sodium pyruvate.

16. The composition of claim 15, wherein said culture medium further comprises 0.05 μ

g/mL to 100 μ

g/mL of putrescine and 1 ng/mL to 100 ng/mL of epidermal growth factor.

17. The composition of claim 14, wherein said composition further comprises a cryopreservative.

18. A purified population of umbilical cord lining stem cells, wherein said cells are positive for CD105, CD106, CD90, CD73, SSEA-4, and STRO-1, negative for CD45, CD34, CD19, and HLA-DR, express OCT4 and Nanog, and do not express Sox2, wherein said cells can undergo at least 60 population doubling in culture.

19. The purified population of ULSCs of claim 18, wherein said cells are capable Of differentiating into cells of mesodermal lineage.

20. The purified population of ULSCs of claim 19, wherein said cells are capable of differentiating into adipogenic cells, osteogenic cells, chondrogenic, and cardiogenic cells.

21. The purified population of ULSCs of claim 18, wherein said cells have undergone at least 70 population doublings in culture.

22. The purified population of ULSCs of claim 18, wherein said cells have undergone at least 90 population doublings in culture.

23. The purified population of ULSCs of claim 18, wherein said cells comprise an exogenous nucleic acid.

24. The purified population of ULSCs of claim 23, wherein said exogenous nucleic acid encodes a polypeptide.

25. The purified population of ULSCs of claim 18, wherein said cells are housed within a scaffold.

26. The purified population of ULSCs of claim 25, wherein said scaffold is biodegradable.

27. The purified population of ULSCs of claim 26, wherein said biodegradable scaffold is composed of collagen.

28. The purified population of ULSCs of claim 18, wherein said cells are capable Of differentiating into cells of the ectodermal lineage.

29. The purified population of ULSCs of claim 28, wherein said cells are capable Of differentiating into neurogenic cells.

30. A method for culturing a population of ULSCs, said method comprising obtaining a population of ULSCs from human umbilical cord, wherein said ULSCs are positive for CD105, CD106, CD90, CD73, SSEA-4, and STRO-1, negative for CD45, CD34, CD19, and HLA-DR, express OCT4 and Nanog, and do not express Sox2;

and culturing said cells in the presence of a low glucose growth medium containing 10% to 20% serum;

0.7 to 1.5% of a stabilized dipeptide of L-alanyl-L-glutamine;

1 to 100 ng/mL of a growth factor selected from the group consisting of bFGF, LIF, and EGF; and

1 to 3% of an antibiotic, wherein said cells can undergo at least 60 population doubling in culture.

31. The method of claim 30, wherein said low glucose growth medium further comprises 0.1 mg/mL to 100 mg/mL of insulin;

0.1 mg/mL to 100 mg/mL of transferrin;

0.1 μ

g/mL to 100 μ

g/mL of selenium; and

0.5 to 1.5% sodium pyruvate.

32. The method of claim 31, wherein said culture medium further comprises 0.05 μ

g/mL to 100 μ

g/mL of putrescine and 1 ng/mL to 100 ng/mL of epidermal growth factor.