Method of partially differentiating hES cells

• US 9,383,349 B2
• Filed: 01/07/2013
• Issued: 07/05/2016
• Est. Priority Date: 03/07/2006
• Status: Active Grant

• Current Assignee: Geeta Shroff
• Original Assignee: Geeta Shroff
• Inventors: Shroff, Geeta
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US13/735,294
• Publication Number:
  US 20130122486A1
• Time in Patent Office: 1,275 Days
• Field of Search: None
• US Class Current: 1/1
• 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: A61K 35/48 Reproductive organs
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 39/00 : General protective or antinoxious agents
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2501/31 : Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
• CPC Class Codes: C12N 2501/392 : Sexual steroids
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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: G01N 33/5044 : involving specific cell types

Abstract

The present invention relates to a pharmaceutical composition comprising of preparations of human embryonic stem (hES) cells and their derivatives and methods for their transplantation into the human body, wherein transplantation results in the clinical reversal of symptoms, cure, stabilization or arrest of degeneration of a wide variety of presently incurable and terminal medical conditions, diseases and disorders. The invention further relates to novel processes of preparing novel stem cell lines which are free of animal products, feeder cells, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor and conditioned media. This invention further relates to the isolation, culture, maintenance, expansion, differentiation, storage, and preservation of such stem cells.

Claims

1. A method of partially differentiating hES cells, free of animal products, feeder cell'"'"'s, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor, and conditioned media, comprising:

(a) introducing expanded hES cells to a cell culture medium consisting of minimal essential medium, wherein the cell culture medium does not contain a progestin or a β

-human chorionic gonadotropin ((β

hCG) agonist; and

(b) incubating the cells at a temperature of about 34 °

C. to about 38 °

C. in an environment of about 3.5% to about 6% carbon dioxide for about 12 hours to about 48 hours in a substantially aerobic environment.

2. The method of claim 1, wherein said cell culture medium is RPMI or DMEM.

3. The method of claim 1, wherein said incubation is carried out in a water jacketed cell culture incubator.

4. The method of claim 1, wherein the cells in (b) are incubated in a biocompatible container.

5. The method of claim 4, wherein said cells differentiate on proliferation.

6. The method of claim 1, further comprising testing the partially differentiated cells for any contamination.

7. The method of claim 1, wherein the ratio of expanded cells to the cell culture medium is about 1:

3.5 to about 1;

35.

8. The method of claim 1, wherein the incubation in (b) is carried out in a biocompatible container and the container is kept in a horizontal position.

9. The method of claim 1, wherein the expanded hES cells in (a) are obtained by a method comprising isolation from a 2 to 7 day old embryo.

10. The method of claim 1, wherein the expanded hES cells in-(a) are obtained by a method comprising isolation from a 2 to 4 day old embryo.

11. The method of claim 1, wherein the expanded hES cells in (a) are obtained by a method comprising isolation from a 2 to 7 day old embryo using mechanical means.

12. The method of claim 1 wherein the expanded hES cells in (a) are obtained by a method comprising:

(i) introducing isolated hES cells in a cell culture medium consisting of minimal essential medium, a progestin, and a β

-human chorionic gonadotropin (β

hCG) agonist; and

(ii) incubating the cells at a temperature of about 34 °

C. to about 38 °

C. in an environment of about 3.5% to about 6% carbon dioxide for about 12 hours to about 48 hours.

13. The method of claim 1, wherein the substantially aerobic environment comprises at least about 15% O₂.

14. The method of claim 1, wherein the expanded hES cells in (a) are obtained by a method comprising resuspension of expanded hES cells comprising:

(A) taking an aliquot of expanded hES cells wherein said aliquot contains at least one stem cell; and

(B) resuspending the cells in cell culture medium comprising progesterone and β

hCG.