Methods of making human primitive ectoderm cells

• US 10,179,902 B2
• Filed: 07/19/2017
• Issued: 01/15/2019
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Agulnick, Alan D., Eliazer, Susan, Kroon, Evert, Baetge, Emmanuel E.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US15/654,624
• Publication Number:
  US 20170313979A1
• Time in Patent Office: 545 Days
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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

This disclosure relates to compositions comprising human preprimitive streak cells and/or human mesendoderm cells as well as methods for their production. Additionally, disclosed herein are methods of identifying factors useful in the further differentiation of preprimitive streak and mesendoderm cell types.

Claims

1. An in vitro method of producing human primitive ectoderm cells, comprising the steps of:

(a) providing a cell population comprising human pluripotent stem cells, and

(b) culturing the human pluripotent stern cells with a(least 100 ng/ml activin A to promote differentiation of the human pluripotent stem cells to human primitive ectoderm cells, said human primitive ectoderm cells are multipotent cells that can differentiate into human mesoderm or human definitive endoderm cells;

thereby producing human primitive ectoderm cells.

2. The method of claim 1, wherein the human pluripotent stem cells are further cultured with Nodal or activin B.

3. The method of claim 1, wherein the human pluripotent stem cells comprise human embryonic stem cells.

4. The method of claim 1, wherein the human pluripotent stem cells are derived from a morula, an inner cell mass (ICM) of an embryo or gonadal ridges of an embryo.

7. The method of claim 1, wherein the population of human primitive ectoderm cells express FZD10, FGF5 or OCT4.

8. The method of claim 1, wherein at least 15% of the pluripotent human stem cells differentiate into the human primitive ectoderm cells.

9. The method of claim 1, wherein at least 50% of the pluripotent human stem cells differentiate into the human primitive ectoderm cells.

10. The method of claim 1, wherein at least 90% of the pluripotent human stem cells differentiate into the human primitive ectoderm cells.

5. The method of claim h further comprising the step of providing serum to the human pluripotent stem cells.

6. The method of claim h further comprising providing a medium that comprises less than 2% (v/v) serum to the human pluripotent stem cells.

11. An in vitro method of producing a cell population comprising at least 10% human primitive ectoderm cells, comprising the steps of:

(a) providing a cell population comprising human pluripotent stem cells, and

(b) culturing the human pluripotent stem cells with at least 100 ng/ml activin A to promote differentiation of the human pluripotent stem cells to human primitive ectoderm cells, said human primitive ectoderm cells are multipotent cells that can differentiate into human mesoderm or human definitive endoderm cells;

thereby generating the cell population comprising at least 10% human primitive ectoderm cells.

12. The method of claim 11, wherein the cell population is cultured in a medium that does not comprise serum.

13. The method of claim 11, wherein the cell population is cultured in a medium that does not comprise serum replacement.