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

• US 20050015824A1
• Filed: 11/12/2002
• Published: 01/20/2005
• Est. Priority Date: 11/09/2001
• Status: Active Grant

• 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.
• Granted Patent: US 7,704,736 B2
• Time in Patent Office: Days
• US Class Current: 800/21
• 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 germ cell specific marker comprising a germ cell specific promoter sequence optionally being operably linked to a reporter gene.

2. The germ cell specific marker of claim 1 comprising a truncated Oct4 promoter gene sequence.

3. The germ cell specific marker of claim 2 comprising the sequence of SEQ ID NO:

1.

4. The germ cells specific marker of claim 3 further comprising a reporter gene.

5. The germ cell specific marker of claim 3 wherein the reporter gene is selected from the group consisting of green fluorescent protein, beta-galactosidase, alkaline phosphatase, luciferase and chloramphenicol acetyltransferase.

6. A method for deriving germ cells from embryonic stem cells comprising:

(a) transforming 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) detaching the cultured cells;

(d) replating the detached cells in medium which prevents further differentiation of said cells;

(e) monitoring the replated cells for reporter gene expression thereby identifying germ cells in a mixed cell population; and

(f) isolating the germ cells identified from step e).

7. The method of claim 6, wherein said germ cells are isolated via a method selected from the group consisting of flow cytometry, enzymatic digestion, and microdissection.

8. The method of claim 6, wherein said differentiation in step b) is promoted by culturing said cells in the absence of leukemia inhibitory factor (LIF).

9. The method of claim 6, wherein said differentiation in step d) is prevented by culturing said cells in the presence of LIF, basic fibroblast growth factor (b-FGF) and stem cell factor.

10. The method of claim 6, optionally further comprising subjecting said cells to culture conditions which promote the formation of oocyte structures.

11. The method of claim 10, said oocyte structures expressing zona pellucida protein 3 (ZP3).

12. The method of claim 6, optionally further comprising subjecting the cells to culture conditions which promote the formation of spermatogonia.

13. The method of claim 12, said spermatogonia expressing at least one specific marker from the group of Scyp1, Protamine 1, and Protamine 2.

14. A germ cell obtained by the method of claim 6.

15. An oocyte obtained via the method of claim 10.

16. Spermatogonia obtained via the method of claim 12.

17. The method of claim 6, wherein said reporter gene is selected from the group consisting of green fluorescent protein, beta-galactosidase, alkaline phosphatase, luciferase and chloramphenicol acetyltransferase

39. A method for preparing a non-human embryo capable of developing into a live-born animal, comprising:

a) obtaining an oocyte as set forth in claim 10;

b) transferring a somatic cell nucleus into said oocyte after enucleating the same to form a chimeric oocyte; and

c) culturing said chimeric oocyte under conditions which result in the establishment of a non-human embryo.

40. The method of claim 39, further comprising implanting said embryo into a recipient female so as to produce a fetus that undergoes full fetal development and parturition to generate said live born animal.

41. An animal obtained via the method of claim 40.

42. The method of claim 39, further comprising the step of transforming said somatic cell nucleus with a corrective nucleic acid sequence prior to transferring said nucleus into said enucleated oocyte.

43. A method for generating genetically identical oocytes from an infertile female, comprising:

a) obtaining an oocyte as set forth in claim 10;

b) transferring a somatic cell nucleus from said infertile female into said oocyte after enucleating the same to form a chimeric oocyte;

c) culturing said oocyte under conditions suitable to induce blastocyst formation;

d) isolating embryonic stem cells from said blastocyst;

e) deriving germ cells from said stem cells; and

f) culturing said germ cells under conditions that promote the formation of oocytes, and

g) isolating the oocytes from step f), said oocytes being genetically identical to said infertile female.

44. An oocyte generated from the method of claim 43.

45. A method for therapeutic cloning of tissues for the treatment of disease in a patient, said method comprising:

a) obtaining an oocyte as claimed in claim 10;

b) transferring a somatic cell nucleus from said patient into said oocyte after enucleating the same to form a chimeric oocyte;

c) culturing said oocyte under conditions suitable to induce blastocyst formation;

d) isolating embryonic stem cells from said blastocyst;

e) exposing said cells to a receptor ligand cocktail which induces the stem cells to differentiate into a desired cell type;

f) culturing the cells of step e) for a suitable time period to generate an effective amount of cells of said desired cell type; and

g) optionally isolating the cells of step f).

46. The method of claim 45, wherein said patient has a neuronal degenerative disease and said desired cell type comprises neurons.

47. The method of claim 45, wherein said patient has heart disease and said desired cell type comprises cardiomyocytes.

48. The method of claim 45, wherein said patient has diabetes and said desired cell type comprises insulin producing islet cells.

49. The method of claim 45, wherein the stem cells of step d) are optionally transformed with a corrective nucleic acid sequence.

50. A method for therapeutic cloning of tissues for the treatment of disease in a patient, said method comprising:

a) obtaining an oocyte as claimed in claim 10;

b) transferring a somatic cell nucleus from said patient into said oocyte after enucleating the same to form a chimeric oocyte;

c) culturing said oocyte under conditions suitable to induce blastocyst formation;

d) isolating embryonic stem cells from said blastocyst;

e) transforming said stem cells with a nucleic acid construct comprising a tissue specific molecule operably linked to a sequence encoding a selectable marker;

f) culturing said cells in the presence of selection agent, those cells comprising the construct surviving in the presence of said selection agent, thereby generating a culture of the desired cell type;

g) culturing the cells of step f) for a suitable time period to generate an effective amount of cells of said desired cell type; and

h) optionally isolating the cells of step g).

51. The method of claim 50, wherein said patient has a neuronal degenerative disease and said tissue specific molecule is nuclear receptor related protein 1 which is expressed in neuronal cells.

52. The method of claim 50, wherein said patient has heart disease and said tissue specific molecule comprises the alpha cardiac myosin heavy chain promoter which is active in cardiomyocytes.

53. The method of claim 50, wherein said patient has diabetes and said tissue specific molecule comprises the human insulin regulatory region which is active in insulin producing islet cells.

54. The method of claim 50, wherein said stem cells of step d) are optionally transformed with a corrective nucleic acid sequence.

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

a) providing an oocyte as claimed in claim 10;

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.

18. A method for deriving germ cells from embryonic stem cells comprising:

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

(b) detaching the cultured cells;

(c) replating the detached cells in medium which prevents further differentiation of said cells;

(d) monitoring the replated cells for morphological alterations associated with germ cell formation; and

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

19. The method of claim 18, wherein said morphological changes comprise at least one of the following:

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

20. The method of claim 18, wherein said germ cells are isolated via a method selected from the group consisting of flow cytometry, enzymatic digestion, and microdissection.

21. The method of claim 18, wherein said differentiation in step a) is promoted by culturing said cells in the absence of leukemia inhibitory factor (LIF).

22. The method of claim 18, wherein said differentiation in step c) is prevented by culturing said cells in the presence of LIF, basic fibroblast growth factor (b-FGF) and stem cell factor.

23. The method of claim 18, optionally further comprising subjecting said cells to culture conditions which promote the formation of oocyte structures.

24. The method of claim 23, said oocyte structures expressing zona pellucida protein 3 (ZP3).

25. The method of claim 18, optionally further comprising subjecting the cells to culture conditions which promote the formation of spermatogonia.

26. The method of claim 25, said spermatogonia expressing at least one specific marker from the group of Scyp1, Protamine 1, and Protamine 2.

27. A germ cell obtained by the method of claim 18.

28. An oocyte obtained via the method of claim 23.

29. Spermatogonia obtained via the method of claim 25.

30. A method for deriving germ cells from embryoid bodies comprising:

(a) transforming embryonic stem cells with a germ cell specific marker comprising a truncated Oct4 gene promoter linked to a reporter gene;

(b) culturing cells comprising the germ cell specific marker under conditions which promote the formation of embryoid bodies;

(c) monitoring the cultured embryoid bodies for germ cell specific reporter gene expression; and

(d) isolating germ cells from the cultured embryoid bodies.

31. The method of claim 30, wherein embryoid body formation is promoted by culturing the cells via the hanging drop method for 48 hours.

32. The method of claim 30, optionally further comprising subjecting said cells to culture conditions which promote the formation of oocyte structures.

33. The method of claim 32, said oocyte structures expressing zona pellucida protein 3 (ZP3).

34. The method of claim 30, optionally further comprising subjecting the cells to culture conditions which promote the formation of spermatogonia.

35. The method of claim 34, said spermatogonia expressing at least one specific marker from the group of Scyp1, Protamine 1, and Protamine 2.

36. A germ cell obtained by the method of claim 30.

37. An oocyte obtained via the method of claim 32.

38. Spermatogonia obtained via the method of claim 34.