Methods for Controlling Stem Cell Differentiation

• US 20090215872A1
• Filed: 06/30/2006
• Published: 08/27/2009
• Est. Priority Date: 07/07/2005
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lee, Jeannie T.
• Application Number:
  US11/988,321
• Publication Number:
  US 20090215872A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/10 naturally occurring
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Disclosed herein are methods for controlling stem cell differentiation through the introduction of transgenes having Xic, Tsix, or Xite sequences to block differentiation and the removal of the transgenes to allow differentiation. Also disclosed are small RNA molecules and methods for using the small RNA molecules to control stem cell differentiation. Also disclosed are stem cells genetically modified by the introduction of Xic, Tsix, or Xite sequences.

Claims

1. A method for delaying differentiation of a stem cell, said method comprising introducing into said stem cell a transgene selected from the group consisting of an Xic transgene, a Tsix transgene, an Xite transgene, a Tsix/Xite transgene, and fragments thereof.

2. The method of claim 1, wherein said Xic transgene comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

1, 2, and 3.

3. The method of claim 1, wherein said Xic transgene comprises a nucleic acid sequence substantially identical to the human Xic sequence set forth in SEQ ID NO:

39.

4. The method of claim 1, wherein said Tsix transgene comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

5, 6, 9, 10, 12, 13, 14, 21, 22, 23, and 28-32.

5. The method of claim 4, wherein said Tsix transgene comprises the nucleic acid sequence of SEQ ID NOs:

9, 10, 12, 21, 22, or 28-32.

6. The method of claim 4, wherein said Tsix transgene comprises at least one copy of the sequence set forth in SEQ ID NOs:

13, 14, or 28-32.

7. The method of claim 6, wherein said Tsix transgene comprises at least two copies of the sequence set forth in SEQ ID NOs:

13, 14, or 28-32.

8. The method of claim 1, wherein said Tsix transgene comprises a nucleic acid sequence substantially identical to the human Tsix sequences set forth in SEQ ID NO:

36 or 40.

9. The method of claim 8, wherein said Tsix transgene comprises at least two copies of the sequence set forth in SEQ ID NO:

40.

10. The method of claim 1, wherein said Xite transgene comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

15, 16, 17, 24, 25, 26, and 27.

11. The method of claim 1, wherein said Xite transgene comprises a nucleic acid sequence substantially identical to the human Xite sequence set forth in SEQ ID NO:

38.

12. The method of claim 1, wherein said Tsix/Xite transgene comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

4, 11, and 19.

13. The method of claim 1, wherein said Tsix/Xite transgene comprises a nucleic acid sequence substantially identical to the human Tsix/Xite sequence set forth in SEQ ID NO:

37.

14. The method of claim 1, wherein said transgene can block endogenous X-X pairing.

15. The method of claim 1, wherein said stem cell is an embryonic stem cell.

16. The method of claim 15, wherein said embryonic stem cell is female or male.

17. The method of claim 15, wherein said embryonic stem cell is mammalian.

18. The method of claim 17, wherein said embryonic stem cell is human or mouse.

20. The method of claim 15, wherein said embryonic stem cell is a blastocyst stage stem cell, an embryonic germ cell, or a cloned stem cell from a somatic nuclei.

21. The method of claim 15, wherein said embryonic stem cell is from an agricultural animal.

19. (canceled)

22. A method for delaying differentiation of a stem cell, said method comprising introducing into said stem cell a small RNA substantially identical to or complementary to at least 15 nucleotides of a transgene selected from the group consisting of an Xic transgene, a Tsix transgene, an Xite transgene, a Tsix/Xite transgene, an Xist transgene, and fragments thereof.

23-31. -31. (canceled)

32. A method of controlling differentiation of a stem cell, said method comprising

(a) introducing into said stem cell at least one transgene selected from the group consisting of an Xic transgene, a Tsix transgene, an Xite transgene, a Tsix/Xite transgene, and fragments thereof, thereby delaying differentiation of said stem cell;

and

(b) when desired, inactivating the transgene, thereby allowing differentiation of said stem cell.

45. The method of claim 32, wherein the transgene further comprises a selectable marker.

46. The method of claim 32, wherein the transgene is flanked by recombinase recognition sequences.

48. The method of claim 32, wherein said inactivating comprises removing said transgene from said stem cell.

49. The method of claim 48, wherein said transgene is removed from said stem cell by expression in said stem cell of a recombinase.

53. The method of claim 32, wherein said stem cell is an embryonic stem cell.

33-44. -44. (canceled)

47. (canceled)

50-52. -52. (canceled)

54-59. -59. (canceled)

60. A stem cell comprising one or more of the following:

an Xic transgene substantially identical to a sequence selected from the group consisting of SEQ ID NOs;

1, 2, 3, 39, and fragments thereof;

a Tsix transgene substantially identical to a sequence selected from the group consisting of SEQ ID NOs;

5, 6, 9, 10, 12, 13, 14, 21, 22, 23, 28-32, 36, 40, and fragments thereof;

an Xite transgene substantially identical to a sequence selected from the group consisting of SEQ ID NOs;

15, 16, 17, 24, 25, 26, 27, 38, and fragments thereof; and

Tsix/Xite transgene substantially identical to a sequence selected from the group consisting of SEQ ID NOs;

4, 11, 19, 37, and fragments thereof.

61-75. -75. (canceled)

76. A isolated small RNA molecule comprising a nucleic acid sequence substantially identical to or complementary to at least 15 nucleotides of a transgene selected from the group consisting of an Xic transgene, a Tsix transgene, an Xite transgene, a Tsix/Xite transgene, an Xist transgene, and fragments thereof.

86. A composition comprising the isolated small RNA molecule of claim 76, formulated to facilitate entry of the small RNA molecule into a cell.

87. A pharmaceutical composition comprising the isolated small RNA molecule of claim 76.

88. A vector comprising the isolated small RNA of claim 76, wherein said small RNA is operably linked to one or more transcriptional regulatory sequences.

77-85. -85. (canceled)