Meningeal-derived stem cells

• US 20040014211A1
• Filed: 06/10/2003
• Published: 01/22/2004
• Est. Priority Date: 06/11/2002
• Status: Active Grant

• Current Assignee: Roy C. Ogle, Sunil Tholpady
• Original Assignee: Roy C. Ogle, Sunil Tholpady
• Inventors: Ogle, Roy C., Tholpady, Sunil
• Granted Patent: US 7,410,797 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0668 : Mesenchymal stem cells from other natural sources

Abstract

Described herein are stem cells derived from the meninges; specifically, the dura mater, pia mater or arachnoid mater. Methods for isolating, differentiating and explanting these cells are described, as well. In particular embodiments, the stem cells of the present invention are differentiated into nerve cells, bone cells, cartilage cells and Schwann cells. The stem cells of the invention can be taken from a small biopsy, and rapidly expanded to large populations of cells using specially defined media that maintain their undifferentiated state. Use of the stem cells of the present invention in biomedical applications is also described.

Claims

1. A stem cell line, comprising cells derived from a meningeal tissue.

2. The stem cell line of claim 1, wherein said meningeal tissue is selected from the group consisting of dura mater, pia mater, arachnoid mater and combinations thereof.

3. The stem cell line of claim 1, wherein said meningeal tissue is obtained by biopsy from a patient or aseptically from a fetus.

4. A composition comprising a substantially pure population of meningeal-derived stem cells.

5. The composition of claim 4, wherein said population of meningeal-derived stem cells includes greater than 80% of totipotent or pluripotent meningeal-derived stem cells.

6. The composition of claim 4, wherein said population of meningeal-derived stem cells includes greater than 90% of totipotent or pluripotent meningeal-derived stem cells.

7. The composition of claim 4, wherein said population of meningeal-derived stem cells includes greater than 99% of totipotent or pluripotent meningeal-derived stem cells.

8. The composition of claim 4, wherein greater than 60% of said population of meningeal-derived stem cells is induced to form a differentiated cell type selected from the group consisting of nerve cell, bone cell, cartilage cell, Schwann cell, adipocyte, fibroblast and melanocyte.

9. The composition of claim 8, wherein said differentiated cell type is nerve cell, and wherein said population of meningeal-derived stem cells is induced to form nerve cells with a nerve cell inducing agent selected from the group consisting of an antioxidant treatment, a steroid hormone or substitute steroid hormone treatment, an agent that stimulates intracellular cyclic AMP and combinations thereof.

10. The composition of claim 9, wherein said nerve cell inducing agent is an antioxidant treatment including an antioxidant selected from the group consisting of β

-mercaptoethanol, butylated hydroxyanisole (BHA), dithiothreitol (DTT), dithioerythritol, tributylphosphine, iodoacetamide, tris-phosphine HCl, deoxythymidine-triphosphate trilithium salt, diethylthiatricarbocyanine perchlorate, diethylthiatricarbocyanine iodide and combinations thereof.

11. The composition of claim 9, wherein said nerve cell inducing agent is a steroid hormone or substitute steroid hormone treatment selected from the group consisting of dexamethasone, pregnenolone, aldosterone, testosterone, estradiol, cortisol, vitamin A and its derivatives, 1,25-dihydroxy vitamin D₃ and combinations thereof.

12. The composition of claim 8, wherein said differentiated cell type is bone cell, and wherein said population of meningeal-derived stem cells is induced to form bone cells with a bone cell inducing agent including laminin.

13. The composition of claim 8, wherein said differentiated cell type is bone cell, and wherein said population of meningeal-derived stem cells is induced to form bone cells with a bone cell inducing agent selected from the group consisting of organic phosphates, inorganic phosphates, ascorbic acid and combinations thereof.

14. The composition of claim 8, wherein said differentiated cell type is cartilage cell, and wherein said population of meningeal-derived stem cells is induced to form cartilage cells with a cartilage cell inducing agent selected from the group consisting of an insulin-selenium-transferrin composition, transforming growth factor (TGF)-β

1 and combinations thereof.

15. The composition of claim 8, wherein said differentiated cell type is Schwann cell, and wherein said population of meningeal-derived stem cells is induced to form Schwann cells with a Schwann cell inducing treatment regimen, comprising:

administering a first treatment, including Dulbecco'"'"'s modified eagle medium (DMEM) and basal medium eagle (BME); and

administering a second treatment, including DMEM, fetal bovine serum (FBS) and retinoid acid.

16. The composition of claim 15, wherein said treatment regimen further includes:

administering a third treatment, including DMEM, FBS, forskolin (FSK), heregulin (HER), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF).

17. The composition of claim 16, wherein said first treatment is administered for about one day, said second treatment is administered for about three days and said third treatment is administered for about five days.

18. The composition of claim 8, wherein said differentiated cell type is Schwann cell, and wherein said population of meningeal-derived stem cells is induced to form Schwann cells with a Schwann cell inducing agent that stimulates intracellular cyclic AMP.

19. The composition of claim 8, wherein said differentiated cell type is adipocyte, and wherein said population of meningeal-derived stem cells is induced to form adipocytes with an adipocyte inducing agent including dexamethasone, insulin, indomethacin and isobutyl-methylxanthine (IBMX).

20. The composition of claim 8, wherein said differentiated cell type is fibroblast, and wherein said population of meningeal-derived stem cells is induced to form fibroblasts with a fibroblast inducing agent including ascorbic acid.

21. The composition of claim 4, further comprising a pharmaceutically acceptable carrier.

22. A method for differentiating a meningeal-derived stem cell into a nerve cell, comprising:

selecting a nerve cell inducing agent from the group consisting of an antioxidant, a steroid hormone, a substitute steroid hormone, an agent that stimulates intracellular cyclic AMP and combinations thereof; and

exposing said meningeal-derived stem cell to a neuronal induction media including said nerve cell inducing agent.

23. The method of claim 22, wherein prior to exposing said meningeal-derived stem cell to said neuronal induction media, said method further includes:

exposing said meningeal-derived stem cell to a neuronal pre-induction media including said nerve cell inducing agent.

24. The method of claim 22, wherein said nerve cell inducing agent is selected from the group consisting of β

-mercaptoethanol, butylated hydroxyanisole (BHA), dithiothreitol (DTT), dithioerythritol, tributylphosphine, iodoacetamide, tris-phosphine HCl, deoxythymidine-triphosphate trilithium salt, diethylthiatricarbocyanine perchlorate, diethylthiatricarbocyanine iodide, dexamethasone, pregnenolone, aldosterone, testosterone, estradiol, cortisol, vitamin A and its derivatives, 1,25-dihydroxy vitamin D₃ and combinations thereof.

25. A method for differentiating a meningeal-derived stem cell into a bone cell, comprising:

selecting a bone cell inducing agent from the group consisting of a laminin substrate, organic phosphates, inorganic phosphates, ascorbic acid and combinations thereof; and

exposing said meningeal-derived stem cell to said bone cell inducing agent.

26. The method of claim 25, wherein said bone cell inducing agent is said laminin substrate, and exposing said meningeal-derived stem cell to said bone cell inducing agent further comprises plating said meningeal-derived stem cell on said bone cell inducing agent.

27. A method for differentiating a meningeal-derived stem cell into a cartilage cell, comprising:

selecting a cartilage cell inducing agent from the group consisting of an insulin-selenium-transferrin (ITS) composition, transforming growth factor (TGF)-β

1 and combinations thereof; and

exposing said meningeal-derived stem cell to said cartilage cell inducing agent.

28. A method for differentiating a meningeal-derived stem cell into a Schwann cell, comprising:

administering a first treatment, including Dulbecco'"'"'s modified eagle medium (DMEM) and basal medium eagle (BME); and

administering a second treatment, including DMEM, fetal bovine serum (FBS) and retinoid acid.

29. The method of claim 28, further comprising:

administering a third treatment, including DMEM, FBS, forskolin (FSK), heregulin (HER), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF).

30. The method of claim 28, wherein said first treatment is administered for about one day, said second treatment is administered for about three days and said third treatment is administered for about five days.

31. A method for differentiating a meningeal-derived stem cell into an adipocyte, comprising:

selecting an adipocyte inducing agent including dexamethasone, insulin, indomethacin and isobutyl-methylxanthine (IBMX); and

exposing said meningeal-derived stem cell to said adipocyte inducing agent.

32. A method for differentiating a meningeal-derived stem cell into a fibroblast, comprising:

selecting a fibroblast inducing agent including ascorbic acid; and

exposing said meningeal-derived stem cell to said fibroblast inducing agent.

33. A method of isolating meningeal-derived stem cells, comprising:

obtaining meningeal tissue from a subject;

washing said meningeal tissue in a physiologic buffer to produce washed meningeal tissue;

placing said washed meningeal tissue in a solution including said physiologic buffer and collagenase to produce dissociated meningeal tissue;

recovering said dissociated meningeal tissue; and

plating said dissociated meningeal tissue onto a culture substrate including a growth medium to culture said meningeal-derived stem cells.

34. The method of claim 33, wherein obtaining said meningeal tissue is performed by a technique selected from biopsy from said subject and aseptic removal from said subject.

35. The method of claim 33, wherein said physiologic buffer is selected from the group consisting of phosphate buffered saline (PBS), Hanks balanced salt solution and combinations thereof.

36. The method of claim 33, wherein placing said washed meningeal tissue in said solution further includes maintaining said washed meningeal tissue in said solution for about 5 to about 30 minutes at a temperature of about 37°

C.

37. The method of claim 33, wherein recovering said dissociated meningeal tissue further includes centrifuging said meningeal tissue and washing a resulting pellet.

38. The method of claim 33, wherein said culture substrate is selected from the group consisting of a tissue culture plate plastic, a laminin-covered substrate, a polyamino acid, fibronectin and type I collagen.

39. The method of claim 33, wherein said growth medium includes Dulbecco'"'"'s modified eagle medium (DMEM), about 10% fetal bovine serum (FBS) and about 1% glutamine.

40. A method of isolating meningeal-derived stem cells, comprising:

obtaining meningeal tissue from a subject;

washing said meningeal tissue in a physiologic buffer to produce washed meningeal tissue;

placing said washed meningeal tissue on a culture substrate including a growth medium to culture said meningeal-derived stem cells.

41. The method of claim 40, wherein obtaining said meningeal tissue is performed by a technique selected from biopsy from said subject and aseptic removal from said subject.

42. The method of claim 40, wherein said physiologic buffer is selected from the group consisting of phosphate buffered saline (PBS), Hanks balanced salt solution and combinations thereof.

43. The method of claim 40, wherein said culture substrate is selected from the group consisting of a tissue culture plate plastic, a laminin-covered substrate, a polyamino acid, fibronectin and type I collagen.

44. The method of claim 40, wherein said growth medium includes Dulbecco'"'"'s modified eagle medium (DMEM), about 10% fetal bovine serum (FBS) and about 1% glutamine.

45. A method of delivering a gene to a recipient, comprising:

inserting a gene construct into a meningeal-derived stem cell; and

implanting said meningeal-derived stem cell into said recipient.