Generation of an autologous stem cell library from human oocytes parthenogenetically activated by high or low oxygen tension

• US 8,420,393 B2
• Filed: 01/17/2012
• Issued: 04/16/2013
• Est. Priority Date: 10/21/2005
• Status: Active Grant

• Current Assignee: International Stem Cell Corporation
• Original Assignee: International Stem Cell Corporation
• Inventors: Revazova, Elena S., Pryzhkova, Marina V., Kuzmichev, Leonid N., Janus, Jeffrey D.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/352,252
• Publication Number:
  US 20120184466A1
• Time in Patent Office: 455 Days
• Field of Search: None
• US Class Current: 435/366
• 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 for generating a library of stem cells comprising autologous stem cells as library members, wherein the stem cells are derived from parthenogenetically activated oocytes from one or more human donors, wherein each library member is homozygous for one or more genes selected from HLA DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 haplotype combinations, the method 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 a library of human stem cells.

2. The method of claim 1, wherein each library member is identified as a full sibling, half sibling, or unrelated to somatic cells of the donor according to single nucleotide polymorphism (SNP) markers.

3. The method of claim 1, wherein the oocyte donor is histocompatible with a member of the library.

4. The method of claim 1, wherein a member of the library is genomically imprinted according to the oocyte donor origin.

5. The method of claim 1, wherein each library member is homozygous for at least one MHC allele present in a human population.

6. The method of claim 1, wherein each library member is homozygous for a different combination of MHC alleles than the other members of the library.

7. The method of claim 1, wherein each library member is at least homozygous for one or more HLA class I genes and HLA class II genes.

8. The method of claim 7, wherein the HLA class I genes are selected from HLA A, HLA B, and HLA Cw haplotype combinations.

9. The method of claim 1, wherein each library member (i) will proliferate in an in vitro culture for over one year, (ii) maintains the potential to differentiate to derivatives of one or all of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iii) is inhibited from differentiation when cultured on a fibroblast feeder layer.

10. The method of claim 9, wherein each library member maintains a karyotype in which the chromosomes are euploid and not altered through prolonged culture.

11. The method of claim 1, wherein each library member can differentiate into to ectoderm, mesoderm, and endoderm germ layers.