Defined media for expansion and maintenance of pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9434920-B2
Application number	US-201313787173-A
Country	US
Kind code	B2
Filing date	Mar 6, 2013
Priority date	Mar 7, 2012
Publication date	Sep 6, 2016
Grant date	Sep 6, 2016

Abstract

The present invention provides methods to promote the proliferation of undifferentiated pluripotent stem cells in defined media. Specifically, the invention provides a defined cell culture formulation for the culture, maintenance, and expansion of pluripotent stem cells, wherein culturing stem cells in the defined cell culture formulation maintains the pluripotency and karyotypic stability of the cells for at least 10 passages. Further disclosed is a cell population grown under defined media conditions that express OCT4, SOX2, NANOG, and FOXA2.

First claim

What is claimed is:

1. A method for the expansion of human pluripotent stem cells comprising culturing the human pluripotent stem cells on a feeder-free matrix in a defined cell culture formulation to thereby expand the cells,
wherein the defined cell culture formulation consists essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, from about 5 ng/ml to about 10 ng/ml of a TGF-β ligand, from about 50 ng/ml to about 100 ng/ml of bFGF, insulin growth factor 1 (IGF-1) and ascorbic acid,
wherein culturing the human pluripotent stem cells in the defined cell culture formulation maintains the pluripotency and karyotypic stability of the cells for at least 10 passages,
and wherein at least 80% of the cells express CD9 at five passages beyond passage 15.

2. The method of claim 1 , wherein the TGF-β ligand is TGF-β1.

3. The method of claim 1 , wherein the fatty acid free albumin is reagent grade.

4. The method of claim 1 , wherein the defined culture formulation consists essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, from about 0.2% to about 2.5% of fatty-acid free albumin, from about 5 ng/ml to about 10 ng/ml of a TGF-β ligand, from about 50 ng/ml to about 100 ng/ml of bFGF, from about 10 ng/ml to about 50 ng/ml of IGF-1 and from about 0.2 mM to about 0.3 mM of ascorbic acid.

5. The method of claim 1 , wherein the defined culture formulation consists of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, from about 5 ng/ml to about 10 ng/ml of a TGF-β ligand, from about 50 ng/ml to about 100 ng/ml of bFGF, insulin growth factor 1 (IGF-1) and ascorbic acid.

6. The method of claim 1 , wherein the defined culture formulation consists of DMEM-F12 basal medium, insulin, transferrin, selenium, from about 0.2% to about 2.5% of fatty-acid free albumin, from about 5 ng/ml to about 10 ng/ml of a TGF-β ligand, from about 50 ng/ml to about 100 ng/ml of bFGF, from about 10 ng/ml to about 50 ng/ml of IGF-1 and from about 0.2 mM to about 0.3 mM of ascorbic acid.

Assignees

• Janssen Biotech Inc

Inventors

• Rezania Alireza

Classifications

• C12N2500/38

  Vitamins · CPC title

• C12N2500/36

  Lipids · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N2501/105

  Insulin-like growth factors [IGF] · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title