Compositions for the derivation of germ cells from stem cells and methods of use thereof

• US 7,704,736 B2
• Filed: 11/12/2002
• Issued: 04/27/2010
• Est. Priority Date: 11/09/2001
• Status: Expired due to Term

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Huebner, Karin M., Scholer, Hans R.
• Primary Examiner(s): Hama; Joanne
• Application Number:
  US10/494,795
• Publication Number:
  US 20050015824A1
• Time in Patent Office: 2,723 Days
• Field of Search: 435/325, 435/377
• US Class Current: 435/377
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 15/877 Techniques for producing new mammalian cloned embryos
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 5/0609 Oocytes, oogonia fertilised oocytes C12N5/0604
• CPC Class Codes: G01N 33/5014 for testing toxicity

Abstract

Compositions and methods are provided for the reproducible derivation of germ cells and oocytes and spermatogonia therefrom. Also provide are methods of use of the same in reproductive and therapeutic cloning protocols.

Claims

1. A method for deriving oocytes from mouse embryonic stem (ES) cells comprising:

(a) transforming mouse embryonic stem cells with a germ cell specific promoter sequence operably linked to a reporter gene;

(b) culturing the transformed cells in ES cell medium under conditions which promote differentiation;

(c) optionally removing the differentiated cells from step b) from said conditions which promote differentiation;

(d) monitoring the cells of step (b) or (c) for reporter gene expression thereby identifying germ cells in a mixed cell population; and

(e) isolating oocytes from the germ cells identified from step (d);

wherein said reporter gene encodes a fluorescent protein and

wherein said germ cell specific promoter sequence is a truncated Oct4 promoter.

2. The method of claim 1, wherein said oocytes are isolated via a method selected from the group consisting of flow cytometry, enzymatic digestion, and microdissection.

3. The method of claim 1, wherein said differentiation in step b) is promoted by culturing said cells in the absence of leukemia inhibitory factor (LIE).

4. The method of claim 1, wherein said removing of cultured cells from said conditions which promote differentiation in step (c) comprises replating said cultured cells in media comprising LIE, basic fibroblast growth factor (b-EGF) and stem cell factor.

5. The method of claim 1, optionally further comprising subjecting said germ cells identified in step d) to culture conditions which promote the formation of oocyte structures.

6. The method of claim 5, wherein said oocyte structures express zona pellucida protein 3 (ZP3).

7. The method of claim 1, wherein said reporter gene is green fluorescent protein.

8. The method of claim 1, wherein said truncated Oct4 promoter gene sequence is an Oct4 promoter gene sequence with conserved regions 2 and 3 deleted.

9. The method of claim 8, wherein said truncated Oct4 promoter gene sequence is SEQ ID NO:

1.

10. An oocyte obtained by the method of claim 1, wherein said truncated Oct4 promoter gene sequence is an Oct4 promoter gene sequence which lacks conserved regions 2 and 3.

11. An oocyte obtained via the method of claim 5, wherein said truncated Oct4 promoter gene sequence is an Oct4 promoter gene sequence which lacks conserved regions 2 and 3.

19. A method for screening test compounds for toxicity or teratogenic potential comprising:

a) providing an oocyte as claimed in claim 5;

b) exposing said oocytes to increasing amounts of said test compound; and

c) culturing said oocytes under conditions that promote blastocyst formation; and

d) determining the effect, if any, of said test compound on the ability of said oocyte to form a blastocyst.

12. A method for deriving oocytes from mouse embryonic stem (ES) cells comprising:

(a) culturing the mouse embryonic stem cells in ES cell medium under conditions which promote differentiation;

(b) optionally removing the differentiated cells from step a) from said conditions which promote differentiation;

(c) monitoring the cells of step (a) or (b) for morphological alterations associated with germ cell formation; and

(d) isolating oocytes from the germ cells identified from the morphological alterations observed in step (c).

13. The method of claim 12, wherein said morphological alterations comprise at least one of the following:

cell enlargement, increased spherical appearance, and reduced cell to cell contacts.

14. The method of claim 12, wherein said oocytes are isolated via a method selected from the group consisting of flow cytometry, enzymatic digestion, and microdissection.

15. The method of claim 12, wherein said differentiation in step a) is promoted by culturing said cells in the absence of leukemia inhibitory factor (LIE).

16. The method of claim 12, wherein said removing of cultured cells from said conditions which promote differentiation in step (b) comprises replating said cultured cells in media comprising LIE, basic fibroblast growth factor (b-FGF) and stem cell factor.

17. The method of claim 12, optionally further comprising subjecting said germ cells identified in step c) to culture conditions which promote the formation of oocyte structures.

18. The method of claim 17, wherein said oocyte structures express zona pellucida protein 3 (ZP3).