Preprimitive streak and mesendoderm cells

• US 9,732,318 B2
• Filed: 07/25/2011
• Issued: 08/15/2017
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan D., D'Amour, Kevin Allen, Eliazar, Susan, Kroon, Evert, Baetge, Emmanuel E.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US13/190,390
• Publication Number:
  US 20120021511A1
• Time in Patent Office: 2,213 Days
• Field of Search: 435366, 435377
• 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 mesendoderm cells, the method comprising the steps of:

(a) obtaining a cell population comprising human pluripotent stem cells;

(b) providing the human pluripotent stem cells with at least one TGFβ

superfamily growth factor in an amount sufficient to promote differentiation of the human pluripotent stem cells to mesendoderm cells, said mesendoderm cells being multipotent cells that can differentiate into mesoderm or definitive endoderm cells; and

(c) allowing sufficient time for the mesendoderm cells to form, wherein the sufficient time for the mesendoderm cells to form has been determined by detecting the presence of mesendoderm cells in the cell population.

2. The method of claim 1, wherein, the TGFβ

superfamily growth factor is selected from the group consisting of Nodal, activin A and activin B.

3. The method of claim 2, wherein the TGFβ

superfamily growth factor is activin A.

4. The method of claim 3, wherein at least 10 ng/ml activin A is provided.

5. The method of claim 3, wherein at least 100 ng/ml activin A is provided.

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

7. The method of claim 1, wherein the human pluripotent stem cells are derived from a tissue selected from the group consisting of the morula, the ICM of an embryo and the gonadal ridges of an embryo.

8. The method of claim 1 further comprising the step of providing serum to the human pluripotent stem cells.

9. The method of claim 1 further comprising a medium that comprises less than about 2% (v/v) serum.

10. The method of claim 1, wherein the population of mesendoderm cells express at least one marker selected from the group consisting of brachyury, FGF4 or SNAI1 and at least one marker from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 or SOX1.

11. The method of claim 10, wherein the expression of the marker selected from the group consisting of brachyury, FGF4 and/or SNAI1 is greater than the expression of the marker selected from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 and/or SOX1 in the population of mesendoderm cells.

12. The method of claim 1, wherein at least about 15% of the pluripotent human stem cells differentiate into mesendoderm cells.

13. The method of claim 1, wherein at least about 50% of the pluripotent human stem cells differentiate into mesendoderm cells.

14. The method of claim 1, wherein at least about 90% of the pluripotent human stem cells differentiate into mesendoderm cells.

15. The method of claim 1, wherein the said pluripotent human stem cells are further provided with one or more growth factors selected from the group consisting of FGF10, FGF4, FGF2 or Wnt3B.

18. The method of claim 1, further comprising a medium that comprises no serum.

19. The method of claim 18, further comprising a medium that comprises no serum replacement.

20. The method of claim 2, further comprising a medium that comprises no serum.

16. An in vitro method of producing human mesendoderm cells, the method comprising:

obtaining a cell population comprising human pluripotent stem cells; and

providing said cell population with a TGFβ

superfamily growth factor, thereby generating a cell population comprising at least 10% mesendoderm cells.

17. The method of claim 16, further comprising allowing sufficient time for the population of mesendoderm cells to form.