Differentiation of stem cells

• US 20060068496A1
• Filed: 07/29/2005
• Published: 03/30/2006
• Est. Priority Date: 07/29/2004
• Status: Abandoned Application

• Current Assignee: Amphioxus Cell Technologies, Inc. (Stem Cell Innovations, Inc.)
• Original Assignee: Amphioxus Cell Technologies, Inc. (Stem Cell Innovations, Inc.)
• Inventors: Kelly, James H.
• Application Number:
  US11/194,143
• Publication Number:
  US 20060068496A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]

Abstract

Disclosed are compositions and methods for identifying specific cell types.

Claims

1. A pluripotent stem cell containing a nucleic acid segment, wherein the nucleic acid segment comprises the structure P-I, wherein P is a transcriptional control element and I is a sequence encoding a marker, wherein the marker comprises a transformation agent.

2. The stem cell of claim 1, wherein the nucleic acid segment is a heterologous nucleic acid segment.

3. The stem cell of claim 1, wherein the nucleic acid segment is an exogenous nucleic acid segment.

4. The stem cell of claim 1, wherein the marker is heterologous.

5. The stem cells of claim 1, wherein P and I are contained in the same vector.

6. The stem cells of claim 1, wherein P and I are contained in different vectors.

7. The stem cell of claim 1, wherein I is a heterologous nucleic acid sequence.

8. The stem cell of claim 7, wherein the nucleic acid segment further comprises a suicide gene.

9. The stem cell of claim 7, wherein P is a tissue specific transcriptional control element.

10. The stem cell of claim 7, wherein P is a cell type specific transcriptional control element.

11. The stem cell of claim 7, wherein P is a cell lineage specific transcriptional control element.

12. The stem cell of claim 7, wherein P is a cell specific transcriptional control element.

13. The stem cell of claim 7, wherein P causes I to be preferentially or selectively expressed.

14. The stem cell of claim 7, wherein the marker comprises a temperature permissive immortalization agent.

15. The stem cell of claim 7, wherein the transformation agent is a temperature permissive agent.

16. The stem cell of claim 7, wherein I comprises the SV40 large T antigen.

17. The stem cell of claim 7, wherein the nucleic acid segment is flanked by a site-specific excision sequence.

18. The stem cell of claim 7, wherein I is flanked by a site-specific excision sequence.

19. The stem cell of claim 7, wherein P is flanked by a site-specific excision sequence.

20. The stem cell of claim 7, wherein the nucleic acid segment further comprises X, wherein X is a site-specific excision sequence, wherein X flanks P-I, wherein the nucleic acid segment comprises the structure X-P-I-X.

21. The stem cell of claim 20, wherein the nucleic acid segment is excised at X.

22. The stem cell of claim 21, wherein X is a loxP site.

23. A differentiated cell produced by culturing the stem cell of claim 7 under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed.

24. The differentiated cell of claim 23, wherein the conditions in which the transcriptional control element is activated are conditions in which the stem cell differentiates.

25. The differentiated cell of claim 23, wherein the stem cell differentiates under the conditions in which the transcriptional control element is activated.

26. The differentiated cell of claim 23, wherein the transcriptional control element is activated by allowing the stem cells to spontaneously differentiate into an embryoid body.

27. The differentiated cell of claim 23, wherein the nucleic acid segment is excised from the differentiated cell.

28. The differentiated cell of claim 27, wherein the nucleic acid segment is excised using an adenovirus-mediated site-specific excision.

29. The differentiated cell of claim 27, wherein the nucleic acid segment is excised using a recombinase.

30. The differentiated cell of claim 29, wherein the recombinase is Cre.

31. The differentiated cell of claim 27, wherein the excision of the nucleic acid segment results in recombination of the nucleic acid molecule from which the nucleic acid segment is excised.

32. The differentiated cell of claim 23, wherein the effect of the expression of I is reversed.

33. The differentiated cell of claim 32, wherein the effect of expression of I is transformation of the differentiated cell, wherein reversal of the effect of the expression of I is reversal of transformation of the differentiated cell.

34. The differentiated cell of claim 32, wherein the effect of the expression of I is reversed by expression of a dominant negative transformation agent.

35. The differentiated cell of claim 32, wherein the effect of the expression of I is reversed by excision of the nucleic acid segment.

36. The differentiated cell of claim 23, wherein the differentiated cell is a hepatocyte.

37. The differentiated cell of claim 23, wherein the differentiated cell is a stem cell derived conditionally immortal cell.

38. A method comprising introducing the differentiated cell of claim 23 into a subject.

39. The method of claim 38, wherein the differentiated cell is introduced by administering the differentiated cell to the subject.

40. The method of claim 38, wherein the differentiated cell is introduced by transplanting the differentiated cell into the subject.

41. A method of assaying a composition for toxicity, the method comprising incubating the composition with the differentiated cell of claim 23, and assessing the differentiated cell for toxic effects.

42. A method of assaying a compound for toxicity, the method comprising incubating the compound with the differentiated cell of claim 23, and assessing the differentiated cell for toxic effects.

43. A method of assaying a composition for an effect of interest on a cell, the method comprising incubating the composition with the differentiated cell of claim 23, and assessing the differentiated cell for the effect of interest.

44. A method of assaying a compound for an effect of interest on a cell, the method comprising incubating the compound with the differentiated cell of claim 23, and assessing the differentiated cell for the effect of interest.

45. A method of deriving differentiated cells from stem cells, the method comprising:

culturing the stem cells of claim 7 under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed, thereby deriving differentiated cells.

46. A method of deriving stem cell derived conditionally immortal cell types, the method comprising:

culturing the stem cells of claim 7 under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed, thereby deriving stem cell derived conditionally immortal cell types.

47. A method of deriving stem cell derived conditionally immortal cell types, the method comprising:

transfecting stem cells with a nucleic acid segment comprising the structure P-I, wherein P is a transcriptional control element and I is a sequence encoding a marker, wherein the marker comprises a transformation agent;

culturing the stem cells under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed, thereby deriving stem cell derived conditionally immortal cell types.

48. A method of deriving differentiated cells from stem cells, the method comprising:

transfecting stem cells with a nucleic acid segment comprising the structure P-I, wherein P is a transcriptional control element and I is a sequence encoding a marker, wherein the marker comprises a transformation agent;

culturing the stem cells under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed, thereby deriving differentiated cells.

49. The method of claim 48, wherein the conditions in which the transcriptional control element is activated are conditions in which the stem cells differentiate.

50. The method of claim 48, wherein the stem cells differentiate under the conditions in which the transcriptional control element is activated.

51. The method of claim 48, wherein the transcriptional control element is activated by allowing the stem cells to spontaneously differentiate into an embryoid body.

52. The method of claim 48 further comprising selecting cells expressing I.

53. The method of claim 48 further comprising increasing the purity of the cells expressing I.

54. The method of claim 53, wherein increasing the purity comprises creating a clonal or semi-purified population of cells.

55. The method of claim 48 further comprising excising the nucleic acid segment.

56. The method of claim 48 further comprising cloning the differentiated cells.

57. The method of claim 48 further comprising culturing the differentiated cells.

58. The method of claim 48 further comprising freezing the differentiated cells.

59. The method of claim 48 further comprising adding a gene of interest to the selected cells.

60. The method of claim 48 further comprising:

excising the nucleic acid segment; and

freezing of the selected cells.

61. The method of claim 60, wherein the ends of the nucleic acid formerly containing the nucleic acid segment recombine when the nucleic acid segment is excised.

62. The method of claim 48 further comprising culturing the cells expressing I.

63. The method of claim 62, further comprising cloning the cultured cells expressing I.

64. The method of claim 48 further comprising introducing the differentiated cells into a subject.

65. The method of claim 64, wherein the differentiated cell is introduced by administering the differentiated cell to the subject.

66. The method of claim 64, wherein the differentiated cell is introduced by transplanting the differentiated cell into the subject.

67. The method of claim 48 further comprising incubating a composition with the differentiated cells, and assessing the differentiated cells for toxic effects.

68. The method of claim 48 further comprising incubating a compound with the differentiated cells, and assessing the differentiated cells for toxic effects.

69. The method of claim 48 further comprising incubating a composition with the differentiated cells, and assessing the differentiated cells for an effect of interest.

70. The method of claim 48 further comprising incubating a compound with the differentiated cells, and assessing the differentiated cells for an effect of interest.

71. A method of deriving differentiated cells from stem cells, the method comprising:

transfecting stem cells with a nucleic acid segment comprising the structure P-I, wherein P is a transcriptional control element and I is a sequence encoding a marker;

culturing the stem cells under conditions in which the transcriptional control element is activated, whereby I is preferentially or selectively expressed, wherein the conditions in which the transcriptional control element is activated are conditions in which the stem cells differentiate thereby deriving differentiated cells.

72. The method of claim 71 further comprising selecting the differentiated cells by selecting for the marker.

73. The method of claim 71 further comprising screening for the differentiated cells be identifying cells expressing the marker.

74. The method of claim 71, wherein the stem cells differentiate under the conditions in which the transcriptional control element is activated.

75. The method of claim 71, wherein the transcriptional control element is activated by allowing the stem cells to spontaneously differentiate into an embryoid body.