Efficient induction of definitive endoderm from pluripotent stem cells

• US 10,487,313 B2
• Filed: 09/15/2017
• Issued: 11/26/2019
• Est. Priority Date: 06/21/2011
• Status: Active Grant

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Funa, Nina, Hess, Katja, Ekberg, Jenny, Semb, Henrik
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US15/705,705
• Publication Number:
  US 20180002668A1
• Time in Patent Office: 802 Days
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention relates to a method to differentiate pluripotent stem cells to a primitive streak cell population, in a stepwise manner for further maturation to definitive endoderm.

Claims

1. A method for differentiating human embryonic stern cells into definitive endoderm cells, comprising:

(a) incubating human embryonic stem cells in a first medium comprising CHIR in a range of 2-7 μ

M, wherein activin A is not present in the medium; and

(b) subsequently incubating the stem cells in a second medium comprising activin A, wherein CHIR is not present in the second medium; and

wherein human definitive endoderm cells are obtained.

2. The method according to claim 1, wherein the concentration of CHIR is at least about 3 μ

M.

3. The method according to claim 1, wherein the concentration of CHIR is about 4 μ

M.

4. The method according to claim 1, wherein the incubating step with CHIR is performed for at least 24 hours.

5. The method according to claim 1, wherein the incubating step with CHIR is performed for 24 hours.

6. The method according to claim 1, wherein the incubating step with CHIR is performed for between 24 and 48 hours.

7. The method according to claim 1, wherein the concentration of activin A is selected from the group consisting of about 80 ng/ml and about 100 ng/ml.

8. The method according to claim 1, wherein the concentration of activin A is 100 ng/ml.

9. The method according to claim 1, wherein the incubating step with activin A is performed for 48 to 72 hours.

10. The method according to claim 2, wherein at least 91.5% of the stem cells after step (b) are positive for SOX17.

11. A method for differentiating human embryonic stem cells into definitive endoderm cells, comprising:

(a) incubating human embryonic stem cells in a first medium comprising about 2-7 μ

M CHIR for 24 hours, wherein activin A is not present in the first medium; and

(b) subsequently incubating the stem cells in a second medium comprising activin A in a concentration of about 100 ng/ml for 48 to 72 hours, wherein CHIR is not present in the second medium, wherein at least 91.5% of the stem cells after step (b) are positive for SOX17; and

wherein human definitive endoderm cells are obtained.

12. The method according to claim 11, wherein the concentration of CHIR is 4 μ

M and the concentration of activin A is 100 ng/ml.