Anterior endoderm cells and methods of production

• US 10,465,162 B2
• Filed: 10/31/2018
• Issued: 11/05/2019
• Est. Priority Date: 04/27/2004
• 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, Baetge, Emmanuel E.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US16/177,235
• Publication Number:
  US 20190055514A1
• Time in Patent Office: 370 Days
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• 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

Disclosed herein are cell cultures comprising PDX1-positive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified PDX1-positive endoderm cells as well as methods for enriching, isolating and purifying PDX1-positive endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of endoderm cells, such as PDX1-positive foregut endoderm cells and PDX1-negative definitive endoderm cells, are also disclosed.

Claims

1. An in vitro method for producing human anterior foregut endoderm cells, comprising:

differentiating human pluripotent stem cells to definitive endoderm cells, and

contacting the definitive endoderm cells with an effective amount of a retinoid to produce human anterior foregut endoderm cells.

2. The in vitro method of claim 1, wherein the retinoid is retinoic acid (RA).

3. The in vitro method of claim 2, wherein the RA is provided to the population of human definitive endoderm cells at a concentration of about 1 μ

M.

4. The in vitro method of claim 1, wherein the human anterior foregut endoderm cells express HOXA3.

5. An in vitro method for producing human midgut endoderm cells, comprising:

differentiating human pluripotent stem cells to definitive endoderm cells, and

contacting the definitive endoderm cells with an effective amount of a retinoid to produce human midgut endoderm cells.

6. The in vitro method of claim 5, wherein the retinoid is RA.

7. The in vitro method of claim 6, wherein the RA is provided to the population of human definitive endoderm cells at a concentration of about 0.2 μ

M.

8. An in vitro method for producing human hindgut endoderm cells, comprising:

differentiating human pluripotent stem cells to definitive endoderm cells, and

contacting the definitive endoderm cells with an effective amount of a retinoid to produce human hindgut endoderm cells.

9. The in vitro method of claim 8, wherein the retinoid is RA.

10. The in vitro method of claim 9, wherein the RA is provided to the population of human definitive endoderm cells at a concentration of at least 0.04 μ

M.

11. An in vitro method for producing anterior foregut endoderm cells, midgut endoderm cells and/or hindgut endoderm cells, comprising:

differentiating human pluripotent stem cells to definitive endoderm cells, and

contacting the definitive endoderm cell population with a retinoid in an amount sufficient to produce the human anterior foregut endoderm cells, midgut endoderm cells and/or human hindgut endoderm cells.

12. The in vitro method of claim 11, wherein the retinoid is RA.

13. The in vitro method of claim 12, wherein the RA is provided to the human definitive endoderm cell population at a concentration from about 0.04 μ

M to about 1 μ

M.

14. The in vitro method of claim 12, wherein the RA is provided to the human definitive endoderm cell population at concentration of about 0.04 μ

M and human hindgut endoderm cells are produced.

15. The in vitro method of claim 12, wherein the RA is provided to the human definitive endoderm cell population at a concentration of about 0.2 μ

M and human midgut endoderm cells are produced.

16. The in vitro method of claim 12, wherein the RA is provided to the human definitive endoderm cell population at a concentration of about 1 μ

M and human anterior foregut endoderm cells are produced.

17. The in vitro method of claim 14, wherein the human hindgut endoderm cells express HOXA13.

18. The in vitro method of claim 15, wherein the human midgut endoderm cells express HOXC6.

19. The in vitro method of claim 15, wherein the human midgut endoderm cells express CDX1.

20. The in vitro method of claim 16, wherein the human anterior foregut endoderm cells express HOXA3.