Proliferation and differentiation of stem cells using extracellular matrix and other molecules

• US 20030113812A1
• Filed: 09/30/2002
• Published: 06/19/2003
• Est. Priority Date: 10/02/2001
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Hemperly, John J.
• Application Number:
  US10/259,815
• Publication Number:
  US 20030113812A1
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/70 Polysaccharides
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: G01N 33/5029 on cell motility
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

Methods and compositions for testing agents for their effects on growth and differentiation of cells, primarily stem cells of various origin, are disclosed. Also disclosed are methods for inducing growth and differentiation of bone marrow stem cells primarily along the pathway to neuronal progenitor cells.

Claims

1. A method for evaluating a candidate agent for its cell growth-inducing or cell differentiation-inducing activity, comprising:

(a) forming a support surface coated with a cell-adhesion resistant (CAR) material and binding thereto at least one bioactive agent, resulting in a surface with at least one exposed CAR region and at least one exposed bioactive region, wherein said CAR material, said at least one bioactive agent or a combination of both are the candidate agents;

(b) adding cells to said support surface such that cells contact each of said regions; and

(c) detecting or measuring cell growth and/or differentiation on each of said regions, wherein the presence of cell growth or differentiation on a region is indicative that said candidate inducing agent present in said region has said growth-inducing or said differentiation-inducing activity.

2. The method of claim 1, wherein said at least one CAR region comprises a CAR material selected from the group consisting of (a) polyethylene glycol, (b) glyme, (c) a glyme derivative, (d) poly-HEMA, (e) polyisopropylacrylamide, (f) hyaluronic acid, (g) alginic acid and (h) a combination of any of (a)-(g).

3. The method of claim 1, wherein said at least one bioactive region comprises a bioactive agent which is an extracellular matrix molecule or a growth factor.

4. The method of claim 3, wherein said bioactive agent is an extracellular matrix molecule selected from the group consisting of laminin, vitronectin, fibronectin, elastin, collagen I, collagen III, collagen IV, collagen VI, entactin, a proteoglycan, and Matrigel™

.

5. The method of claim 1 wherein said bioactive agent is an adhesion domain of an extracellular matrix molecule or other cell adhesion molecule.

6. The method of claim 1, wherein said detecting or measuring comprises visualizing the binding of an antibody to said cells.

7. The method of claim 1, wherein said at least one CAR region comprises a CAR material bonded to a tissue culture-treated surface.

8. The method of claim 1, wherein said at least one CAR material is bonded to the surface through an intermediate layer which is bonded directly to said surface.

9. The method of claim 8, wherein said intermediate layer comprises polyethyleneimine, poly-L-lysine, poly-D-lysine, poly-L-ornithine, poly-D-ornithine, poly(vinylamine), or poly(allylamine).

10. The method of claim 1 or 2, wherein said CAR material is oxidized with a mild oxidizing agent prior to adding said bioactive agent.

11. The method of claim 10 wherein said oxidizing agent is sodium periodate.

12. The method of claim 3, wherein said bioactive region comprises growth factors.

13. The method of claim 1, wherein said at least one bioactive region is in the form of a plurality of spots, with each spot comprising at least one type of bioactive molecule deposited thereon.

14. The method of claim 1, wherein said at least one bioactive region is in the form of a plurality of spots, with each spot comprising a different concentration of one type of bioactive molecule.

15. The method of claim 1, wherein said at least one bioactive region is in the form of spots in a grid pattern on said surface.

16. The method of claim 13, wherein said plurality of spots are in a grid pattern on said surface.

17. The method of claim 14, wherein said plurality of spots are in a grid pattern on said surface.

18. The method of claim 13 wherein said bioactive molecule is an extracellular matrix molecule.

19. The method of claim 14 wherein said bioactive molecule is an extracellular matrix molecule.

20. The method of claim 1, wherein said at least one bioactive region is in the form of a plurality of wells, with each well comprising at least one type of bioactive molecule deposited thereon.

21. The method of claim 20 wherein said bioactive molecule is an extracellular matrix molecule.

22. The method of claim 1, wherein said cells are stem cells.

23. The method of claim 22, wherein said stem cells are pluripotent stem cells.

24. The method of claim 22, wherein said stem cells are progenitor stem cells.

25. The method of claim 22, wherein said stem cells are embryonic stem cells.

26. The method of claim 22, wherein said stem cells are hematopoietic stem cells.

27. The method of claim 22, wherein said stem cells are bone marrow-derived stem cells.

28. The method of claim 22, wherein said stem cells are osteogenic stem cells.

29. The method of claim 22 wherein said stem cells differentiate into neuronal progenitor cells.

39. The method of claim 28, wherein said bioactive agents are extracellular matrix molecules.

30. A method for inducing or promoting the differentiation of stem cells into neuronal progenitor cells comprising:

(a) contacting stem cells in a culture vessel having a surface coated with one or more cell adhesion resisting (CAR) agents and one or more bioactive agents that induce stem cell differentiation to neural progenitor cells; and

(b) culturing said stem cells for a time sufficient to permit them to differentiate into neuronal progenitor cells.

31. The method of claim 30 further comprising

(c) detecting said differentiation.

32. The method of claim 30 wherein said bioactive agents are extracellular matrix molecules.

33. The method of claim 30 wherein said bioactive agents are a combination of a polycationic polyamino acid and an extracellular matrix molecule.

34. The method of claim 33 wherein said combination is selected from the group consisting of:

(a) poly-omithine and laminin;

(b) poly-omithine and fibronectin;

(c) poly-omithine and collagen VI;

(d) poly-omithine and vitronectin;

(e) poly-lysine and collagen VI;

(f) poly-lysine and vitronectin; and

(g) poly-ornithine and poly-lysine.

38. A method for producing an isolated or enriched population of neuronal progenitor cells from stem cells, comprising:

(a) inducing or promoting the differentiation of stem cells into neuronal progenitor cells in accordance with claim 30, thereby producing said neuronal progenitor cells;

(b) optionally, detecting said differentiation; and

(c) enriching or isolating said neuronal progenitor cells.

42. The method of any of claims 30, 35, 38 or 40 wherein the CAR material is selected from the group consisting of (a) polyethylene glycol, (b) glyme, (c) a glyme derivative, (d) poly-HEMA, (e) polyisopropylacrylamide, (f) hyaluronic acid, (g) alginic acid and (h) a combination of any of (a)-(g).

43. The method of claim 42 wherein said CAR material is hyaluronic acid.

44. The method of any of claims 30, 35, 38 or 40, wherein said CAR material is bonded to an intermediate layer which is bonded to said surface.

45. The method of claim 44 wherein said intermediate layer is selected from the group consisting of polyethyleneimine, poly-L-lysine, poly-D-lysine, poly(vinylamine), and poly(allylamine).

46. The method any of claims 30, 35, 38 or 40 wherein said cells are stem cells.

47. The method claim 46, wherein said stem cells are bone marrow stem cells.

48. The method claim 46, wherein said stem cells are pluripotent stem cells.

49. The method of claim 31 or 36, wherein said detecting step comprises detecting or measuring nestin in or on cells that have differentiated to neuronal progenitor cells.

50. The method of claim 42 wherein nestin is detected or measured using an antibody specific for nestin.

35. A method for inducing or promoting the differentiation of stem cells into neuronal progenitor cells comprising:

(a) contacting stem cells in a culture vessel having a surface with a combination of juxtaposed regions of different bioactive agents or different concentrations of a bioactive agent, which combination is capable of inducing stem cell differentiation to neural progenitor cells; and

(b) culturing said stem cells for a time sufficient to permit them to differentiate into neuronal progenitor cells.

36. The method of claim 35 further comprising

(c) detecting said differentiation.

37. The method of claim 35 wherein said bioactive agents are extracellular matrix molecules.

40. A method for producing an isolated or enriched population of neuronal progenitor cells, comprising:

(a) inducing or promoting the differentiation of stem cells into neuronal progenitor cells in accordance with claim 35, thereby producing said neuronal progenitor cells;

(b) optionally, detecting said differentiation; and

(c) enriching or isolating said neuronal progenitor cells.

41. The method of claim 40, wherein said bioactive agents are extracellular matrix molecules.

51. An article useful for evaluating a candidate agent for its cell growth-inducing or cell differentiation-inducing activity, comprising:

(a) a support surface coated with a cell-adhesion resistant (CAR) material having bound thereto at least one bioactive agent, such that the resulting surface comprises at least one exposed CAR region and at least one exposed bioactive region, wherein said CAR material, said at least one bioactive agent or a combination of both is the candidate agent.

52. The article of claim 51, wherein said at least one CAR region comprises a CAR material selected from the group consisting of (a) polyethylene glycol, (b) glyme, (c) a glyme derivative, (d) poly-HEMA, (e) polyisopropylacrylamide, (f) hyaluronic acid, (g) alginic acid and (h) a combination of any of (a)-(g).

53. The article of claim 51, wherein said at least one CAR region comprises a tissue culture-treated surface to which said CAR material is bonded.

54. The article of claim 51, wherein said at least one bioactive region comprises a bioactive agent which is an extracellular matrix molecule or a growth factor.

55. The article of claim 53, wherein said at least one bioactive region comprises extracellular matrix molecules selected from the group consisting of laminin, vitronectin, fibronectin, elastin, collagen I, collagen III, collagen IV, collagen VI, entactin, a proteoglycan, or Matrigel™

.

56. The article of claim 51, wherein said at least one CAR region is bonded to the surface through an intermediate layer which is bonded directly to said surface.

57. The article of claim 56, wherein said intermediate layer comprises polyethyleneimine, poly-L-lysine, poly-D-lysine, poly(vinylamine), or poly(allylamine).

58. The article of claim 54, wherein said bioactive region comprises growth factors selected from the group consisting of a bone morphogenetic proteins, epidermal growth factor, erythropoietin, heparin binding factor, hepatocyte growth factor, insulin, insulin-like growth factor I or II, an interleukin, a muscle morphogenic protein, nerve growth factor, platelet-derived growth factor, and transforming growth factor α

or β

.

59. The article of claim 51, wherein said at least one bioactive region is in the form of a plurality of spots, with each spot comprising at least one bioactive agent deposited thereon.

60. The article of claim 51, wherein said at least one bioactive region is in the form of a plurality of spots, with each spot comprising a different concentration of one bioactive agent.

61. The article of claim 59 or 60, wherein said plurality of spots are arrayed in a grid pattern on said surface.