Particulate Delivery Vehicles for Embryoid Bodies

• US 20090263359A1
• Filed: 02/04/2009
• Published: 10/22/2009
• Est. Priority Date: 02/04/2008
• Status: Active Grant

• Current Assignee: Children's Hospital Boston, Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Ferreira, Lino da Silva, Kohane, Daniel, Langer, Robert
• Granted Patent: US 8,642,333 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells

Abstract

The present invention provides a vehicle for delivering various chemicals, compositions and proteins to stem cells and embryoid bodies. The vehicle may be biocompatible and biodegradable polymer microparticles. Typically the particles will contain at least a growth factor for delivery to the embryoid bodies, and generally the growth factor induces differentiation of the cells in the embryoid body along a specific lineage. The present invention also provides methods for directing differentiation of the cells in the embryoid body.

Claims

1. A method of directing differentiation of stem cells comprising the steps of:

providing stem cells;

providing a microparticle wherein the microparticle comprises a growth factor;

contacting the stem cells with the microparticles under conditions suitable to effect differentiation of the cells of the embryoid bodies.

2. The method of claim 1 further comprising centrifuging the mixture of cells and microparticles.

3. The method of claim 1 wherein the particles are comprised of a bio-compatible and biodegradable polymer.

4. The method of claim 3 wherein the polymer comprises poly(lactide-co-glycolide) (PLGA).

5. The method of claim 2 wherein the particles are comprised of a material selected from the group comprising poly(anhydrides), poly(hydroxy acids), poly(ortho esters), poly(propylfumerates), poly(caprolactones), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes, poly(glycerol sebacates), elastomeric poly(glycerol sebacates polysaccharides), and combinations thereof.

6. The method of claim 1 wherein the particles are comprised of a material selected from the group comprising polypyrrole, polyanilines, polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonates, poly(ethylene oxide), co-polymers and combinations thereof.

7. The method of claim 1 wherein the cells, after contacting with the microparticles, express CD 34.

8. The method of claim 1 wherein the cells after contacting with the microparticles, express PECAM1.

9. The method of claim 1 wherein the particles have a mean diameter in the range between about 0.2 μ

m and about 25 μ

m.

10. The method of claim 1 wherein the particles comprise a protein.

11. The method of claim 1 wherein the particles comprise a small molecule.

12. The method of claim 1 wherein the particles comprise DNA.

13. The method of claim 1 wherein the particles comprise RNA.

14. The method of claim 13 wherein the particles comprise RNAi.

15. The method of claim 1 wherein the growth factor is selected from the group comprising vascular endothelial growth factor (VEGF165), basic fibroblast growth factor (bFGF), placenta growth factor (P1GF), and combinations thereof.

16. The method of claim 1 wherein the growth factor is selected from the group comprising epidermal growth factor, nerve growth factor, transforming growth factor-β

, platelet-derived growth factor, insulin-like growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, hepatocyte growth factor, brain-derived neurotrophic factor, keratinocyte growth factor, bone morphogenetic protein, a cartilage-derived growth factor, and combinations thereof.

17. The method of claim 1 wherein the stem cells are human stem cells.

18. The method of claim 17 wherein the stem cells are embryonic stem cells.

19. The method of claim 17 wherein the stem cells are adult stem cells.

20. The method of claim 1 wherein the particles may be taken up by the cells of the embryoid bodies.

21. The method of claim 1 wherein the particles are not capable of being taken up by the cells of the embryoid bodies.

22. The method of claim 1 wherein the differentiation of the cells is toward cells of the mesoderm germlayer.

23. The method of claim 1 wherein the differentiation of the cells is toward cells of the endoderm germlayer.

24. The method of claim 1 wherein the differentiation of the cells is toward cells of the ectoderm germlayer.

25. The method of claim 1 wherein the differentiation of the cells is away from cells of the endoderm germlayer.

26. The method of claim 1 wherein the differentiation of the cells is away from cells of the ectoderm germlayer.

27. The method of claim 1 wherein the differentiation of the cells is toward mesenchymal cells.

28. The method of claim 1 wherein the differentiation of the cells is toward endothelial cells.

29. The method of claim 1 wherein the differentiation of the cells is toward vascular cells.

30. The method of claim 1 wherein the differentiation of the cells is toward neuronal cells.

31. The method of claim 1 wherein the differentiation of the cells is toward hepatic cells.

32. The method of claim 1 wherein the first providing step comprises providing about 30,000 stem cells.

33. A composition comprising embryoid bodies, and microparticles wherein the microparticles comprise a polymer, and a growth.

34. The composition of claim 33 further comprising a differentiation medium.

35. The composition of claim 33 wherein the microparticles are comprised of a biocompatible and biodegradable polymer.

36. The composition of claim 35 wherein the polymer comprises poly(lactide-co-glycolide) (PLGA).

37. The composition of claim 33 wherein the microparticles have a mean diameter in the range between about 0.2 μ

m and about 25 μ

m.

38. The composition of claim 33 wherein the microparticles comprise a protein.

39. The composition of claim 33 wherein the microparticles comprise a small molecule.

40. The composition of claim 33 wherein the microparticles comprise DNA.

41. The composition of claim 33 wherein the microparticles comprise RNA.

42. The composition of claim 33 wherein the microparticles comprise RNAi.

43. The composition of claim 33 wherein the microparticles have different release profiles.

44. The composition of claim 33 wherein the microparticles have different degradation profiles.

45. The composition of claim 33 wherein the microparticles are fluorescent.

46. The composition of claim 33 wherein the microparticles are magnetic.

47. The composition of claim 33 wherein the microparticles comprise a plurality of growth factors.

48. The composition of claim 33 wherein the growth factor is one or more, selected from the group comprised of vascular endothelial growth factor (VEGF165), basic fibroblast growth factor (bFGF) and placenta growth factor (P1GF).

49. The composition of claim 33 wherein the particles may be taken up by the cells comprising the embryoid bodies.

50. The composition of claim 33 wherein the particles may not capable of being taken up by the cells comprising the embryoid bodies.

51. The composition of claim 33 wherein the differentiation of the embryoid bodies is toward cells of the mesoderm germ layer.

52. A method of directing differentiation of cells of embryoid bodies comprising the steps of:

providing embryoid bodies;

providing microparticles wherein the microparticles comprise VEGF165, bFGF, or P1GF; and

contacting the embryoid bodies with the microparticles under conditions suitable to effect differentiation of the cells of the embryoid bodies;

wherein the cells of the embryoid bodies differentiate toward vascular cells.