Differentiation of Pluripotent Stem Cells

• US 20110091971A1
• Filed: 06/29/2009
• Published: 04/21/2011
• Est. Priority Date: 06/30/2008
• Status: Abandoned Application

• Current Assignee: Centocor Ortho Biotech Incorporated (Johnson & Johnson)
• Original Assignee: Centocor Ortho Biotech Incorporated (Johnson & Johnson)
• Inventors: Liu, Jiajian, Pardinas, Jose, Chi, Ellen, Hunter, Michael Joseph, Connor, Judith Ann, Davis, Janet, Swanson, Ronald Vernon, Raghunathan, Gopalan
• Application Number:
  US12/493,741
• Publication Number:
  US 20110091971A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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

Abstract

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention is directed to methods and compositions to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage. The present invention also provides methods to generate and purify agents capable of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage.

Claims

1. A method to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, comprising treating the pluripotent stem cells with a medium containing a peptide comprising the amino acid sequence of activin A containing at least one point mutation, for a period of time sufficient for the pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage.

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

3. The method of claim 1, wherein the at least one point mutation is at least one of the amino acid residues in the amino acid sequence of activin A selected from the group consisting of:

10I, 16F, 39Y, 41E, 43E, 74F, 75A, 76N, 77L, 78K, 79S, and 82V.

4. The method of claim 3, wherein the at least one point mutation is selected from the group consisting of:

a deletion, an insertion and a substitution.

5. The method of claim 1, wherein the at least one point mutation at least one of the amino acid residues in the amino acid sequence of activin A selected from the group consisting of:

16F, 18V, 19S, 20F, 37A, 38N, 39Y, 41E, 74F, 82V, 107N, 109I, 110V, and 116S.

6. The method of claim 5, wherein the at least one point mutation is selected from the group consisting of:

a deletion, an insertion and a substitution.

7. The method of claim 1, wherein the peptide comprising the amino acid sequence of activin A containing at least one point mutation is further modified to contain at least one region that is capable of specifically binding to a ligand on a solid substrate in an affinity purification column.

8. The method of claim 7, wherein the at least one region that is capable of specifically binding to a ligand on a solid substrate in an affinity purification column is a metal binding site.

9. The method of claim 8, wherein the metal binding site comprises a pair of histidine residues.