Compositions and methods for neural differentiation of embryonic stem cells

• US 20060121607A1
• Filed: 08/08/2003
• Published: 06/08/2006
• Est. Priority Date: 08/08/2002
• Status: Abandoned Application

• Current Assignee: The University of Georgia Research Foundation, Inc., Bresagen, Inc. (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Bresagen, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Schulz, Thomas, Stice, Steven L., Condie, Brian G., Davidson, Bruce
• Application Number:
  US10/524,157
• Publication Number:
  US 20060121607A1
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The present invention provides compositions and methods for human neural cell production. More particularly, the present invention provides cellular differentiation methods employing an essentially serum free MEDII conditioned medium for the generation of human neural cells from pluripotent and multipotent human stem cells.

Claims

1. A method of producing a human neural cell comprising,

a) providing a pluripotent human cell;

b) forming an embryoid body by contacting the pluripotent human cell with an essentially serum free medium; and

c) culturing the embryoid body in an essentially serum free cell differentiation environment

to thereby produce the human neural cell.

2. The method of claim 1, wherein the essentially serum free medium of step b) comprises Ham'"'"'s F12 medium.

3. The method of claim 1, wherein the essentially serum free medium of step b) comprises a MEDII conditioned medium.

4. The method of claim 1, wherein the essentially serum free cell differentiation environment of step c) comprises a MEDII conditioned medium.

5. The method of claim 1, comprising the additional steps performed after step b) and before step c):

a) dispersing the embryoid body to an essentially single cell suspension;

b) culturing the essentially single cell suspension in a suspension culture; and

c) forming a second embryoid body by culturing the essentially single cell suspension with a second essentially serum free medium, wherein the second essentially serum free medium comprises a MEDII conditioned medium.

6. The method of claim 5, wherein one or more of the essentially serum free media is essentially LIF free.

7. The method of claim 5, wherein the second essentially serum free medium comprises DMEM/F12, FGF-2 and a MEDII conditioned medium.

8. The method of claim 5, wherein the second essentially serum free medium comprises between approximately 10% to approximately 50% MEDII conditioned medium.

9. The method of claim 5, wherein the essentially serum free medium, the second essentially serum free medium, and/or the essentially serum free cell differentiation environment comprises less than 5% serum.

10. The method of claim 5, wherein the cell differentiation environment is selected from the group consisting of adherent culture and suspension culture.

11. The method of claim 1, wherein the human cell is a pluripotent human cell.

12. The method of claim 11, wherein the pluripotent human cell is selected from the group consisting of a human embryonic stem cell, a human inner cell mass (ICM)/epiblast cell, a human primitive ectoderm cell, a human primordial germ cell, a human teratocarcinoma cell.

15. The method of claim 12, wherein the human primitive ectoderm cell is an early primitive ectoderm (EPL) cell.

18. The method of claim 11, wherein the pluripotent human cell is a human embryonic stem cell, and wherein the human embryonic stem cell is passaged by selection with a SSEA4 antibody and/or with a sequential collagenase and trypsin treatment prior to forming an embryoid body.

19. The method of claim 1, wherein the human cell is a multipotent human cell.

20. The method of claim 5, wherein the MEDII conditioned medium is a Hep G2 conditioned medium.

24. The method of claim 5, wherein the MEDII medium comprises a low molecular weight component selected from the group consisting of a proline residue, and a polypeptide comprising one or more proline residues.

26. A neural cell produced by the methods of claim 1.

27. A method for treating a patient, comprising a step of administering to the patient having a neural disease a therapeutically effective amount of the neural cell of claim 26.

28. The method of claim 27, wherein the neural disease is Parkinson'"'"'s disease.

13. (canceled)

14. (canceled)

16. (canceled)

17. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

25. (canceled)

29. A method of producing a partially differentiated pluripotent cell comprising culturing a pluripotent cell culture on a layer of fresh feeder cells, wherein the fresh feeder cells have been plated for less than approximately 48 hours, thereby inducing formation of a more differentiated pluripotent cell.

30. The method of claim 29, wherein the fresh feeder cells have been plated for less than approximately 24 hours.

31. The method of claim 29, wherein the fresh feeder cells have been plated for less than approximately 12 hours.

32. The method of claim 29, wherein the fresh feeder cells have been plated for less than approximately 6 hours.

33. The method of claim 29, wherein the pluripotent cell culture forms a colony after it is cultured on the layer of fresh feeder cells, and the more differentiated pluripotent cell is selected from the central region of the colony.

34. The method of claim 33, wherein the more differentiated pluripotent cell expresses less Oct4 than an embryonic stem cell.

35. The partially differentiated cell generated using the method of claim 29.

36. A neural cell culture composition comprising a population of neural cells derived in vitro from pluripotent cells, wherein the neural cells express one or more detectable markers for tyrosine hydroxylase (TH), vesicular monamine transporter 2 (VMAT2), aromatic amino acid decarboxylase (AADC) and dopamine transporter (DAT).

38. The composition of claim 36, wherein at least one of the cultured cells expresses all of the detectable markers TH, VMAT2, AADC and DAT.

39. The composition of claim 36, wherein the neural cell is a human cell.

37. (canceled)

40. A neural cell culture composition comprising a population of neural cells derived in vitro from pluripotent cells, wherein the neural cells express one or more detectable markers for nestin or vimentin, and the neural cells have the capacity to differentiate into cells of a neural lineage.

42. The neural cell culture composition of claim 40, wherein the neural lineage is selected from the group consisting of neurons, astrocytes, oligodendrocytes and Schwann cells.

44. The neural cell culture composition of claim 40, wherein the cells of the neural lineage express a neurotransmitter phenotype selected from the group consisting of a GABAergic neuron, a cholinergic neuron, a glutamatergic neuron, a glycinergic neuron, a noradrenergic neuron, an adrenergic neuron, a sertonergic neuron, and a histaminergic neuron.

45. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a GABAergic neuron that expresses glutamate decarboxylase and/or expresses vesicular inhibitory amino acid transporter/vesicular gaba transporter.

46. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a cholinergic neuron that expresses choline acetyltransferase and/or vesicular acetylcholine transporter.

47. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a glutamatergic neuron that expresses vesicular glutamate transporter.

48. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a glycinergic neuron that expresses vesicular inhibitory amino acid transporter.

49. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a noradrenergic neuron that expresses norepinephrine transporter.

50. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a adrenergic neuron that expresses phenylmethanolamine N-methyl transferase.

51. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a serotonergic neuron that expresses tryptophan hydroxylase or serotonin transporter.

52. The neural cell culture composition of claim 44, wherein the neurotransmitter phenotype is a histaminergic neuron that expresses histidine decarboxylase.

53. The composition of claim 40, wherein the neural cell is a human cell.

41. (canceled)

43. (canceled)