Methods of making human primitive ectoderm cells

• US 10,550,367 B2
• Filed: 10/02/2018
• Issued: 02/04/2020
• 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:
  US16/149,999
• Publication Number:
  US 20190032008A1
• Time in Patent Office: 490 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 mesoderm cells, comprising:

(a) culturing human pluripotent stem cells in a first medium comprising activin A for about 24 hours;

(b) removing the activin A from the first medium to form a first cell population; and

(c) culturing the first cell population of step (b) in a second medium comprising no activin A for a sufficient time to form a second cell population comprising mesoderm cells.

2. The in vitro method of claim 1, wherein the first medium does not comprise serum.

3. The in vitro method of claim 1, wherein the first medium comprises activin A at a concentration of at least 30 ng/ml.

4. The in vitro method of claim 1, wherein the first medium comprises activin A at a concentration of 100 ng/ml.

5. The in vitro method of claim 1, wherein in step (c) the first cell population is cultured in the second medium for about 48 hours.

6. The in vitro method of claim 1, wherein the mesoderm cells express Brachyury, forkhead box protein (FOXF1), vascular endothelial growth factor receptor 2 (FLK1), bone morphogenetic protein 4 (BMP4), homeobox protein MOX1, or stromal cell derived factor 1 (SDF1).

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

8. The in vitro method of claim 1, wherein the first cell population comprises mesendoderm cells.

9. The in vitro method of claim 1, wherein the second medium comprises at least 0.2% serum.

10. An in vitro method of producing preprimitive streak cells, comprising:

culturing human pluripotent stem cells in a medium comprising activin A for about 6 hours to about 24 hours,

thereby forming preprimitive streak cells; and

isolating the preprimitive streak cells.

11. The in vitro method of claim 10, wherein the medium does not comprise serum.

14. The in vitro method of claim 10, wherein the preprimitive streak cells express fibroblast growth factor 8 (FGF8), nuclear localized β

-catenin, or both FGF8 and nuclear localized β

-catenin.

15. The in vitro method of claim 14, wherein the expression of FGF8 or nuclear localized β

-catenin in the preprimitive streak cells is greater than the expression of brachyury, fibroblast growth factor 4 (FGF4), zinc finger protein SNAI1, SRY-related HMG-box 17 (SOX-17), hepatocyte nuclear factor 3-beta (FOXA2), SRY-related HMG-box 7 (SOX7), or SRY-related HMG-box 1 (SOX1) on the preprimitive streak cells.

16. The in vitro method of claim 10, wherein the human pluripotent stem cells are human embryonic stem cells.

17. The in vitro method of claim 10, wherein the medium comprises about 0.2% serum.

18. The in vitro method of claim 10, wherein the medium comprises about 0.2% serum to about 2.0% serum.

12. An in vitro method of producing preprimitive streak cells, comprising:

culturing human pluripotent stem cells in a medium comprising activin A at concentration of at least 30 ng/ml for about 6 hours to about 24 hours, thereby forming preprimitive streak cells.

13. The in vitro method of claim 12, wherein the medium comprises activin A at a concentration of 100 ng/ml.