Defined media for expansion and maintenance of pluripotent stem cells

• US 9,593,307 B2
• Filed: 05/09/2016
• Issued: 03/14/2017
• Est. Priority Date: 03/07/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Visone, Thomas J
• Application Number:
  US15/150,349
• Publication Number:
  US 20160251615A1
• Time in Patent Office: 309 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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

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 expresses OCT4, SOX2, NANOG, and FOXA2.

Claims

1. A method for expanding human pluripotent stem cells comprising culturing the human pluripotent stem cells on a feeder-free matrix in a defined cell culture formulation,

wherein the defined cell culture formulation consists essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, from about 0.5 ng/ml to about 10 ng/ml of a TGF-β

ligand, from about 50 ng/ml to about 100 ng/ml of bFGF, and from about 10 ng/ml to about 50 ng/ml of IGF-1, and

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.

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

ligand is TGF-β

1.

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

4. The method of claim 1, wherein at least 80% of the cells express CD9 at five passages beyond passage 15.

5. The method of claim 1, wherein the defined culture formulation consists of DMEM F-12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β

ligand, bFGF, and IGF-1.

6. The method of claim 1, wherein the defined culture formulation consists of DMEM F-12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, TGF-β

1, bFGF, and IGF-1.

7. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

8. The method of claim 1, wherein the defined cell culture formulation lacks ascorbic acid.

9. The method of claim 1, wherein the defined cell culture formulation consists essentially of about 1 ng/ml of TGF-β

1.

10. The method of claim 1, wherein the defined cell culture formulation consists essentially of about 100 ng/ml of bFGF.

11. The method of claim 1, wherein the defined cell culture formulation consists essentially of about 20 ng/ml of IGF-1.

12. The method of claim 1, wherein the defined cell culture formulation consists essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, about 1 ng/ml of TGF-β

1, about 100 ng/ml of bFGF, and about 20 ng/ml of IGF-1.

13. The method of claim 1, wherein the defined cell culture formulation consists of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, about 1 ng/ml of TGF-β

1, about 100 ng/ml of bFGF, and about 20 ng/ml of IGF-1.