Oxygen tension for the parthenogenic activation of human oocytes for the production of human embryonic stem cells

• US 7,732,202 B2
• Filed: 08/15/2006
• Issued: 06/08/2010
• Est. Priority Date: 10/21/2005
• Status: Active Grant

• Current Assignee: International Stem Cell Corporation
• Original Assignee: International Stem Cell Corporation
• Inventors: Janus, Jeffrey D., Revazova, Elena S., Kuzmichev, Leonid N., Pryzhkova, Marina V.
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US11/505,260
• Publication Number:
  US 20070141702A1
• Time in Patent Office: 1,393 Days
• Field of Search: None
• US Class Current: 435/373
• CPC Class Codes: A61K 35/15 Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00
• CPC Class Codes: A61K 35/26 Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 35/35 Fat tissue; Adipocytes; Stromal cells; Connective tissues adipose-derived stem cells A61K35/28; collagen A61K38/39
• CPC Class Codes: A61K 35/36 Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells islets of Langerhans A61K35/39
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 : for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/8776 : Primate embryos
• CPC Class Codes: C12N 2500/02 : Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/14 : Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/70 : Enzymes
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/1323 : Adult fibroblasts
• CPC Class Codes: C12N 2517/10 : Conditioning of cells for in vitro fecondation or nuclear transfer
• 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
• CPC Class Codes: C12N 5/0609 : Oocytes, oogonia fertilised oocytes C12N5/0604
• CPC Class Codes: C12N 9/12 : transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12Y 207/11001 : Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase

Abstract

Methods of producing human stem cells are disclosed for parthenogenetically activating human oocytes by manipulation of O₂ tension, including manipulation of Ca²⁺ under high O₂ tension and contacting oocytes with serine threonine kinase inhibitors under low O₂ tension, isolating inner cell masses (ICMs) from the activated oocytes, and culturing the cells of the isolated ICMs under high O₂ tension. Moreover, methods are described for the production of stems cells from activated oocytes in the absence of non-human animal products, including the use of human feeder cells/products for culturing ICM/stem cells. Stem cells produced by the disclosed methods are also described.

Claims

1. A method of producing human stem cells comprising:

a) parthenogenetically activating a human oocyte, wherein activating comprises;

i) contacting the oocyte with an ionophore at high O₂ tension and ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O₂ tension;

b) cultivating the activated oocyte of step (a) at low O₂ tension until blastocyst formation;

c) transferring the blastocyst to a layer of feeder cells, and culturing the transferred blastocyst under high O₂ tension;

d) mechanically isolating an inner cell mass (ICM) from trophectoderm of the blastocyst of step (c); and

e) culturing the cells of the ICM of step (d) on a layer of feeder cells, wherein culturing step (e) is carried out under high O₂ tension, thereby producing human stem cells.

2. The method of claim 1, wherein low O₂ tension is maintained by incubation in a gas mixture environment comprising an O₂ concentration of about 2% O₂ to about 5% O₂.

3. The method of claim 2, wherein the gas mixture environment further comprises about 5% CO₂ and about 90% N₂ to 93% N₂.

4. The method of claim 1, wherein high O₂ tension is maintained by incubation in a gas mixture environment comprising about 5% CO₂ and about 20% O₂.

5. The method of claim 1, wherein the ionophore is selected from the group consisting of ionomycin and A23187.

6. The method of claim 1, wherein the serine-threonine kinase inhibitor is selected from the group consisting of staurosporine, 2-aminopurine, sphingosine, and 6- dimethylaminopurine (DMAP).

7. The method of claim 1, wherein the activating, isolating, and culturing steps are carried out under defined media conditions for therapeutic applications.

8. The method of claim 7, wherein the media comprises human umbilical cord serum.

9. The method of claim 8, wherein the media comprises about 10% human umbilical cord serum.

10. The method of claim 1, wherein the layer of feeder cells comprises human fibroblasts.

11. The method of claim 10, wherein the fibroblasts are postnatal human dermal fibroblasts.

12. The method of claim 10, wherein the feeder cells are inactivated with an antibiotic.

13. The method of claim 12, wherein the antibiotic is mitomycin C.

14. A method of activating a human metaphase II oocyte comprising;

a) incubating a human metaphase II oocyte in in vitro fertilization (IYF) media;

b) incubating the cell of step (a) in IVF media comprising an ionophore;

c) incubating the cell of step (b) in IYF media comprising a serine-threonine kinase inhibitor; and

d) incubating the cells of step (c) in fresh IVF medium until blastocyst formation,

wherein the incubating steps (a) and (b) are carried out under high O₂ tension, and wherein an inner cell mass (ICM) obtained from the blastocyst at step (d) produce culturable stem cells.

15. The method of claim 14, wherein the O₂ tension for incubating steps (c) and (d) is maintained by incubating the cells in a gas mixture environment comprising an O₂ concentration of about 2% O₂ to 5% O₂.

16. The method of claim 15, wherein the gas mixture environment further comprises about 5% CO₂ and about 90% N₂ to 93% N₂.

17. The method of claim 14, further comprising incubating the oocytes with hyaluronidase.

18. The method of claim 14, wherein incubating step (a) is carried out for about 2 hours at about 37°

C.

19. The method of claim 14, wherein incubating step (b) is carried out for about 5 minutes at about 37°

C.

20. The method of claim 14, wherein incubating step (c) is carried out for about 4 hours at about 37°

C.

21. The method of claim 14, wherein incubating step (d) is carried out for about 24 hours at about 37°

C.

22. The method of claim 14, wherein the IVF media is free of non-human products.

23. The method of claim 22, wherein the ionophore is selected from the group consisting of ionomycin and A23187.

24. The method of claim 23, wherein the ionophore is ionomycin.

25. The method of claim 22, wherein the serine-threonine kinase inhibitor is selected from the group consisting of staurosporine, 2-aminopurine, sphingosine, and 6- dimethylaminopurine (DMAP).

26. The method of claim 25, wherein the serine-threonine kinase inhibitor is DMAP.