Stem cell maturation for all tissue lines

• US 20030134422A1
• Filed: 01/16/2003
• Published: 07/17/2003
• Est. Priority Date: 01/16/2002
• Status: Abandoned Application

• Current Assignee: PrimeGen Biotech LLC
• Original Assignee: PrimeGen Biotech LLC
• Inventors: Sayre, Chauncey Bigelow
• Application Number:
  US10/346,816
• Publication Number:
  US 20030134422A1
• Time in Patent Office: Days
• US Class Current: 435/455
• 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: C12M 23/58 Reaction vessels connected in series or in parallel combinations of bioreactors with other apparatus, C12M43/00
• CPC Class Codes: C12M 41/26 of pH
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]

Abstract

The present invention provides a composition and method for making a modified germ cell (MGC) comprising a primordial sex cell, (PSC) or nucleus thereof, combined with an enucleated ovum. The PSC is a spermatogonia or an oogonia from a donor animal or mammal. Similarly, the enucleated ovum is from the same species donor animal or mammal as the donor of the PSC. The present invention also provides a method of maturing the MGC in vitro using a specialized cell culture apparatus or bioreactor chamber. The present invention also provides for a method of screening the MGCs for receptor sites, to determine if they are sufficiently matured to be translocated into a host animal or mammal in vivo and/or tissue in vitro. The present invention also provides for a specialized cell culture chamber or bioreactor chamber to grow, expand, maintain, sustain and mature the MGCs, or other types of cells.

Claims

1. A modified germ cell comprising:

a primordial sex cell (PSC), or nucleus thereof, translocated into an enucleated ovum, wherein the PSC and the ovum are derived from the same species or different species of animal.

2. The modified germ cell according to claim 1 wherein the PSC is a spermatogonium.

3. The modified germ cell according to claim 1 wherein the PSC is an oogonium.

4. The modified germ cell according to claim 1 wherein the animal is selected from the group consisting of rodents, primates, felines, canines, equines, porcine, bovines, and ovine.

5. The modified germ cell according to claim 1 wherein the cell is totipotent.

6. The modified germ cell according to claim 1 wherein the wherein the cell is pluripotent.

7. The modified germ cell according to claim 1 wherein the wherein the cell is multipotent.

8. The modified germ cell according to claim 1 wherein the wherein the cell is bipotent.

9. A modified germ cell comprising:

a spermatogonium, or nucleus thereof, translocated into an enucleated ovum, wherein the spermatogonium and the ovum are derived from the same species of animal.

10. A modified germ cell comprising:

a oogonium, or nucleus thereof, translocated into an enucleated ovum, wherein the oogonium and the ovum are derived from the same species of animal.

32. The method for inducing differentiation of a modified germ cell according to claim 31 wherein the modified germ cells are obtained by preparing them according to any one of claims 10 through 30.

11. A method for preparing a modified germ cell comprising:

(a) obtaining a PSC from a first donor animal;

(b) obtaining an ovum from a second donor animal of the same species as the first donor animal;

(c) enucleating the ovum; and

(d) translocating the PSC, or nucleus thereof, into the enucleated ovum.

12. The method for preparing a modified germ cell according to claim 11 wherein the PSC obtained from the first donor animal is a spermatogonium.

13. The method for preparing a modified germ cell according to claim 11 wherein the PSC obtained from the first donor animal is an oogonium.

14. The method for preparing a modified germ cell according to claim 11, further comprising cryopreserving the modified germ cell.

15. A method for maturing a modified germ cell comprising:

(a) expanding the modified germ cell in a first medium;

(b) placing the modified germ cell into a second medium;

(c) screening for membrane receptors on the modified germ cell;

(d) repeating steps b and c until the number of receptors on the modified germ cell are sufficient to be a primed modified germ cell; and

(e) translocating the primed modified germ cell into the tissue of the animal;

17. The method for maturing the modified germ cell according to claim 15, wherein the second medium is derived from a stage two maturing cell(s) from an animal.

19. The method for maturing the modified germ cell according to claim 15, wherein a second media type is dependent on the stage of maturing cell(s) from an animal.

20. The method for maturing the modified germ cell according to claim 15, wherein the animal is a mammal of the same species as the first donor mammal.

21. The method for maturing the modified germ cell according to claim 15, wherein the animal is a mammal of a different species as the first donor mammal.

16. The method for maturing the modified germ cell according to claim 16, wherein the second medium is derived from a stage one maturing cell(s) from an animal.

22. The method for maturing a modified germ cell comprising:

(a) obtaining a PSC from a first donor mammal;

(b) obtaining an ovum from a second donor animal of the same species as the first donor animal;

(c) enucleating the ovum;

(d) translocating the PSC, or nucleus thereof, into the enucleated ovum;

(e) expanding the modified germ cell in a first medium;

(f) placing the modified germ cell into a second medium;

(g) screening for membrane receptors on the modified germ cells;

(h) repeating steps b and c until the number of receptors on the modified germ cell are sufficient to be a primed modified germ cell; and

(i) translocating the primed modified germ cell into the tissue of the animal.

23. The method for maturing the modified germ cell according to claim 22, wherein the first medium is derived from a stage one maturing cell(s) from an animal of the same species as the first donor animal.

24. The method for maturing the mammalian modified germ cell according to claim 22, wherein the second medium is derived from a stage two maturing cell(s) from an animal of the same species as the first donor animal.

25. The method for maturing the modified germ cell according to claim 22, wherein consecutive media is used from consecutively maturing cell(s) from an animal of the same species as the first donor animal until the modified germ cell has comparable receptors as that of the maturing cell.

26. A method for preparing a modified germ cell comprising:

(a) obtaining a PSC from a first donor mammal;

(b) obtaining an ovum from a second donor mammal of the same species of the first donor mammal;

(c) enucleating the ovum;

(d) fusing the PSC and the enucleated mammalian ovum to form a fused cell; and

(e) activating the fused cell.

27. The method for preparing a modified germ cell according to claim 26, wherein the activating step (e) comprises increasing intracellular levels of divalent cations and reducing phosphorylation of cellular proteins in the modified germ cell.

28. The method for preparing a modified germ cell according to claim 26, wherein the medium comprises antimycotic.

29. The method for preparing a modified germ cell according to claim 26, wherein the medium comprises antibiotic.

30. The method for preparing the modified germ cell according claim 26 comprises using an electrical stimulus.

31. A method for inducing differentiation of a modified germ cell comprising:

(a) obtaining at least one mammalian modified germ cell;

(b) culturing the modified germ cells in the presence of differentiating factors under suitable conditions, and for a time sufficient, to induce the cells to differentiate.

33. The method for inducing differentiation of a modified germ cell according to claim 31 wherein the culturing step is performed in vitro.

34. The method for inducing differentiation of a modified germ cell according to claim 31 wherein the in vitro culture is conducted in a multi-chambered cell culture vessel such that the modified germ cells are isolated and separated from the source of the differentiating factors.

35. The method for inducing differentiation of a modified germ cell stem cell according to claim 31 wherein the source of differentiating factors are derived from cells and tissues.

36. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the culturing step is performed in vivo.

37. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the modified germ cells are induced to differentiate by implanting the modified germ cell into an animal.

38. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the implanted modified germ cells are contained within a semi-permeable cell membrane such that the implanted modified germ cell remain isolated from the animal.

39. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the semi-permeable cell membrane is retrieved by attaching a hollow fiber or thread to the membrane.

40. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the hollow fiber or thread is tagged.

41. The method for inducing differentiation of the modified germ cell according to claim 31 wherein the tag is fluorescent.

42. A cell culture chamber comprising:

(a) at least one isolation chamber;

(b) tubing connecting the chamber; and

(c) at least one pump.

43. A cell culture chamber according to claim 42, wherein there is at least two chambers.

44. A cell culture chamber according to claim 42, wherein the chamber contains sufficient fluid appropriate to grow, expand, maintain, sustain and mature cells in vitro.

45. A cell culture chamber according to claim 42, wherein the fluid is further comprised of fetal blood.

46. A cell culture chamber according to claim 42, wherein the chambers are further comprised of different size fittings.

47. A cell culture chamber according to claim 42, wherein the chambers are connected by tubing via the fittings.

48. A cell culture chamber according to claim 42, wherein the fluid in the chamber flows between the chambers via the tubing and driven by the pumps.

49. A cell culture chamber according to claim 42, wherein the chamber is connected to a carbon dioxide source.

50. A cell culture chamber according to claim 42, wherein the chamber is connected to an oxygen source.

51. A cell culture chamber according to claim 42, wherein the chamber is further comprised of different size fittings.

52. A cell culture chamber according to claim 42, wherein there is a pH sensor detecting the hydrogen content of the fluid in the chamber.

53. A cell culture chamber according to claim 42, wherein the pH sensor is further connected to a pH meter that controls the amount of substance to be injected into the fluid in the chamber.

54. A cell culture chamber according to claim 42, wherein the substance is carbon dioxide.

55. A cell culture chamber according to claim 42, wherein the substance is any other substance which returns the pH of the fluid in the chamber to its desired pH.

56. A cell culture chamber comprising:

(a) at least one isolation chamber;

(b) tubing connecting the chamber;

(c) a carbon dioxide source;

(d) an oxygen source;

(e) a molecular filter; and

(f) at least one pump.

57. A cell culture chamber according to claim 56, wherein there is at least two chambers.

58. A cell culture chamber according to claim 56, wherein the chamber contains sufficient fluid volume appropriate to grow, expand, maintain, sustain and mature cells in vitro.

59. A cell culture chamber according to claim 56, wherein the chambers are further comprised of different size fittings.

60. A cell culture chamber according to claim 56, wherein the chambers are connected by tubing via the fittings.

61. A cell culture chamber according to claim 56, wherein the fluid in the chamber flows between the chambers via the tubing and driven by the pumps.

62. A cell culture chamber according to claim 56, wherein the chamber is further comprised of different size fittings.

63. A cell culture chamber according to claim 56, wherein there is a pH sensor detecting the hydrogen content of the fluid in the chamber.

64. A cell culture chamber according to claim 56, wherein the pH sensor is further connected to a pH meter that controls the amount of substance to be injected into the fluid in the chamber.

65. A cell culture chamber according to claim 56, wherein the substance is carbon dioxide from the carbon dioxide source.

66. A cell culture chamber according to claim 56, wherein the substance is any other substance which returns the pH of the fluid in the chamber to its desired pH.